Ginkgolic acids impairs mitochondrial function by decreasing

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Bioactive Constituents, Metabolites, and Functions

Ginkgolic acids impairs mitochondrial function by decreasing mitochondrial biogenesis and promoting FUNDC1-dependent mitophagy Wenjun Wang, Miaomiao Wang, Yu Ruan, Junyang tan, Hao Wang, Tao yang, Jianshuang Li, and Qinghua Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04178 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 16, 2019

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Journal of Agricultural and Food Chemistry

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Ginkgolic acids impairs mitochondrial function by decreasing

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mitochondrial biogenesis and promoting FUNDC1-dependent

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mitophagy

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Wenjun Wang$,#, Miaomiao Wang§,#, Yu Ruan§, Junyang Tan§, Hao Wang$ , Tao Yang&,

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Jianshuang Li§,* and Qinghua Zhou$,§,*

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$,The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong 510632, China

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§, Biomedical Translational Research Institute, Jinan University, Guangzhou, Guangdong

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510632, China

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&, Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, MI

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49503, USA.

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# These

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*Corresponding Authors

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Jianshuang Li : [email protected]

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Qinghua Zhou: [email protected]

authors contributed equally to this work.

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ACS Paragon Plus Environment


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Abstract

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Ginkgolic acids (GA) are found in the leaves, nuts and testa of Ginkgo biloba and have been

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reported to exhibit anti-tumor, anti-bacterial, and pro-apoptosis acitivities. However, its role

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in mitochondrial function is still unclear. Our previously study showed that genes related to

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the mitochondria present significantly changes in GA-treated mouse bone marrow stromal

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cells. We hypothesize that GA may regulate mitochondrial function. Here, we found that GA

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treatment induced mitochondrial fragmentation, reduced mtDNA copy nubmers and

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mitochondrial protein levels, and impaired mitochondrial ATP production and oxygen

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consumption. The GA induced mitochondrial mass loss maybe due to decreased

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mitochondrial biogenesis. In addition, abolishing autophagy by Atg7 knockout or the

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administration of autophagy inhibitor can restore the GA-induced decrease in mitochondrial

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mass. Furthermore, FUNDC1 knockdown restored the GA-induced changes in mitochondrial

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mass reduction and mitochondrial membrane potential loss. Together, our studies

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demonstrated that GA impaired mitochondrial function by decreasing mitochondrial

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biogenesis and promoting FUNDC1-dependent mitophagy.

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Keywords: Ginkgolic acid, mitochondria, mitophagy, FUNDC1

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Introduction

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Mitochondria are highly dynamic and the main energy-producing organelles in mammalian

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cells, but they also play an important role in cell injury and death by releasing pro-death

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molecules and generating toxic reactive oxygen species1. Mitophagy, a selective type of

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autophagy-dependent degradation of mitochondria, is the major pathway for the removal of

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damaged or no-longer-needed mitochondria in eukaryotic cells in various physiological and

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pathological conditions2. For examples, paternal mitochondria elimination is executed by

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mitophagy, which is necessary for the development in C. elegans and Drosophila3, 4. Defects

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in mitophagy contribute to neurodegenerative diseases, inflammation activation, cancer and

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decreased lifespan5-8. The PINK-Parkin pathway is one of the most well-known mitophagy

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pathways. Under normal conditions, the mitochondrial kinase PINK1 is constantly degraded

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through proteasome pathway. While the mitochondria are damaged, PINK1 is stabilized and

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then recruits the ubiquitin E3 ligase Parkin, leading to the ubiquitination of the outer

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mitochondrial membrane proteins , thus triggering mitophagy9. In addition to PINK-parkin

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dependent mitophagy, BNIP3- and FUNDC1-dependent mitophagy pathways were also found

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in multiple contexts. BNIP3 regulates mitophagy in response to hypoxia and is critical for

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erythroid maturation10. BNIP3 has been reported to bind to and preserve PINK1 from

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degradation, thus promoting mitophagy by recruiting Parkin to the mitochondrial

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membrane11. FUNDC1 is a highly conserved mitochondrial outer-membrane protein

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identified as a mitophagy receptor that directly binds LC3 under hypoxic conditions12, 13. At

3



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present, relative to the well-established PINK-Parkin pathway mediated mitophagy,

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mechanisms of the other mitophagy pathways need to be further defined.

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Ginkgo biloba, known as a “ living gymnosperms fossil”, has been used to treat memory and

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cognitive impairment in Chinese medicine from 2000 years ago14. In addition, ginkgo leaf

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extract is used in cosmetics for its functions for skin benfits, such as potent antioxidant

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protection, skin-soothing effects, and reduced signs of aging15. Moreover, ginkgo nut, is an

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edible delicacy in China, Japan and Korean Peninsula16. Ginkgolic acids (GA) is a natural

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component extracted from the leaves, nuts and testa of Ginkgo biloba and shows a wide range

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of biological activities, including anti-tumor, anti-HIV, anti-bacterial, neurotoxic, pro-

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apoptosis, and pro-autophagy effects17-20. GA has been reported to increase resistance against

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oxidative stress in lens epithelial cells21, and to participate in cancer cell migration and

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invasion22. GA also regulates glucose metabolism, inflammation, and cell death in multiple

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cell lines23-25. Furthermore, GA can directly bind to and inhibit sumoylation E1 (SAE1/SAE2)

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enzyme activity26. In addition, GA has been reported to reduce mitochondrial membrane

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potential and promote mitochondrial damage27, 28.

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Our previously study showed that genes related to the mitochondria present significantly

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changes in GA-treated mouse bone marrow stromal cells29, suggesting GA may play an

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important role in mitochondria. However, the exact role and mechanism of GA regulation of

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mitochondrial function are still unclear. Here, we studied the mitochondrial morphology,

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mitochondrial mass and function, as well as the underlying mechanisms upon GA (15:1)

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treatment in HeLa cells. 4


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Materials and Methods

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Cell culture and treatment

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HeLa were purchased from the Shanghai Cell Bank, Type Culture Collection Committee,

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Chinese Academy of Sciences. ATG7 KO, GFP-Parkin-overexpressing and control HeLa

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(gifts from Dr.Quan Chen, Nankai University) cells were maintained in DMEM (Gibco, New

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York, USA, C11995500BT) supplemented with 10% fetal bovine serum (HyClone, Logan，

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USA, SV30160.03) and 1% penicillin and streptomycin (Gibico, New York, USA, 15140-

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122). All cells were incubated at 37°C under 5% CO2. In the treatment groups, the cells were

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treated with 25 M or 50 M GA (Calbiochem, Darmstadt, Germany, 345887) for 24 h.

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Autophagy measurement

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For autophagy analysis, HeLa cells were transfected with GFP-LC3 for 48 h. Following the

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indicated treatment, the cells were fixed with 4% paraformaldehyde and imaged with a Leica

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TCS SP8 confocal microscope. GFP-LC3 puncta were counted in each cell. At least 200 cells

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were analyzed per treatment. A diffuse distribution of GFP-LC3 was considered to represent

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non-autophagic puncta. For inhibition autophagy, cells were treated with 50 M CQ (Sigma,

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St. Louis, USA, C6628) for 6 h.

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BNIP3/FUNDC1 knockdown

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BNIP3 and FUNDC1 shRNA (shBNIP3: 5’ GCCTCGGTTTCTATTTATAAT 3’/ shFUNDC:

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5’ AAGTGATGACGACTCTTATGA 3’) were inserted into the pLKO.1 vector (a gift from

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Dr. Song Z. Y., Wuhan University), which was then transfected into 293T cells. After 48 h of

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transfection, the virus was collected and used to infect HeLa. Stable BNIP3 or FUNDC1 5



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knockdown cell lines were selected using puromycin (Amresco, Pennsylvania, USA, J593) at

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a concentration of 1 g/ml.

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Immunofluorescent staining

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Cells were fixed with 4% formaldehyde and blocked with 2% BSA (Amresco, Pennsylvania,

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USA, E588). Primary antibodies against HSP60 (Proteintech, Proteintech, Chicago, USA,

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15282-1-AP; 1:500 dilution), cytochrome c (CST, Boston, USA, 4280; 1:500 dilution), LC3B

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(Sigma, St. Louis, USA, L7543; 1:500), and LAMP1 (CST, Boston, USA, 9091; 1:500

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dilution) were applied overnight. The cells were then incubated with an anti-rabbit or anti-

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mouse antibody conjugated with Alexa 488 or Alexa 594 (Jackson ImmunoResearch,

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Lancaster, USA, AB_2337249, AB_2307325). Following by DAPI costaining, the cells were

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imaged with a Leica TCS SP8 confocal microscope. For colocalization quantification, images

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were preprocessed with subtraction of a median filter-processed image and background, and

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then two images were proceeded to the ImageJ plugin JACOP (National Institutes of Health 30.

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Western blotting

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Proteins were isolated in ice-cold RIPA buffer (Beyotime, Shanghai, China, P0013B) with

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proteinase inhibitors, and protein concentrations were determined in BCA assays. Proteins

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were fractionated by SDS-PAGE, electroblotted onto PVDF membrane (Millipore,

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Darmstadt, Germany, IPVH00010) and probed with primary antibodies (Table S1). Protein

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bands detected by the antibodies were visualized by enhanced chemiluminescence (Beyotime,

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Shanghai, China, P0018) and evaluated using Quantity One 1-D Analysis Software (Bio-Rad,

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Hercules, CA). 6


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mtDNA measurements

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Total DNA was isolated by using the Gentra Puregene Cell Kit (QIAGEN, New York, USA,

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158388) according to the manufacturer's instructions. For the measurement of mtDNA copy

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numbers, the qPCR primers employed for mitochondrial tRNALeu (UUR) and nuclear -2-

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microglobulin (B2M) (Table S. 2) were used for the qPCR assay. Data analysis of

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mitochondrial contents was performed according to a previously described method 31.

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RNA isolation and qPCR analysis

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Total RNA was isolated from cultured cells using RNA iso Plus (TaKaRa, Tokyo, Japan,

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9109) as we previously reported32. Total RNA was reverse transcribed into cDNA by using

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the ABScript II cDNA First Strand Synthesis Kit (ABclonal, Wuhan, China, RK20400)

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following the manufacturers' protocol. mRNA levels were quantified in a SYBR Green Select

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Master Mix (ABclonal, Wuhan, China, RK21203) on a CFX96 real-time system (Biorad,

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Hercules, CA) The abundance of specific gene transcripts was assessed by qPCR (primers are

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listed in Table S. 2). Actb was used as the internal control. Relative gene expression was

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expressed as the fold change calculated using the 2-ΔΔCT method.

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RNA-Seq analysis and gene set enrichment analysis

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Total RNA was extracted from the control and GA treated HeLa cells. The RNA was then

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sequenced by the WuXi App Tec RNA-seq service (n = 2). GO analysis was performed using

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DAVID (http://david.abcc.ncifcrf.gov/). Gene expression clustering was analyzed using

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Cluster 3.0 and visualized using Java TreeView. For gene set enrichment analysis, we applied

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GSEA v2.2.0 to various functional and/or characteristic gene signatures. Gene sets were 7



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obtained from the MsigDB database v3.0. Statistical significance was assessed by comparing

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the enrichment score to enrichment results generated from 1000 random permutations of the

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gene set to obtain p values (nominal p value).

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MMP (mitochondrial membrane potential) assay

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The JC-1 probe was used to measure the mitochondrial membrane potential (MMP). Cells

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cultured in 12-well plates, following the indicated treatments were incubated with 500 l of

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JC-1 (Beyotime, Shanghai, China, C2006) at 37°C for 20 min. The cells were then washed

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three times with JC-1 staining buffer and imaged with a Nikon Ti2-U microscope.

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Oxygen consumption rate (OCR) measurements

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OCRs were measured using Seahorse XF96 equipment (Seahorse Bioscience Inc., Santa

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Clara, USA). Briefly, cells were seeded at 8,000 cells per well and treated with normal

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medium or medium containing 50 M GA for 24 h in 80 µl of medium. The cell plates were

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incubated in a CO2-free incubator at 37°C for 1 h before the measurements. Analysis was

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performed using 1 µM oligomycin, 0.5 µM FCCP, and 1 µM rotenone as indicated. The data

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were normalized to the protein levels. ATP-dependent respiration (or oligomycin-sensitive

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respiration) was calculated as the difference in the OCRs before and after the addition of

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oligomycin.

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Analysis of ATP levels

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The ATP levels were determined using an ATP assay kit from Beyotime according to the

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manufacturer’s instructions. Briefly, After cell adherence for 12h, the culture medium were

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changed to DMEM (no glucose, 10% FBS) medium, and GA (50M) or DMSO were added

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for 12h or 24h. Cell were collected in lysis buffer. After centrifugation (12000 × g for 5 min)

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to remove cell debris, the pellets were used to determine protein concentrations via BCA

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assays, and the supernatant was added to the substrate solution for the luciferase assay.

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Luminescence was recorded in an Illuminometer with an integration time of 10 s per well.

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ATP levels were normalized to the protein contents of the samples.

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Statistical analysis

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All experiments were carried out in triplicates. The results are expressed as the mean ±

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standard deviation (SD). The level of statistical significance was set at p < 0.05 using an

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unpaired two-tailed Student’s t-test. All statistical analyses were performed using GraphPad

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Prism software.

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Results

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GA treatment regulated mitochondrion related cellular processes

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To examine whether GA regulates mitochondion- related gene expression in a cell type-

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specific manner, we performed RNA sequencing (RNA-Seq) analysis in GA- and DMSO-

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treated HeLa cell. Gene Ontology (GO) analysis showed that mitochondrion- related

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biological processes were significantly changed in the GA- treated group (Fig. 1A-B); the

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identified terms included mitochondrial electron transport-ubiquinol to cytochrome c,

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mitochondrial respirator chain complex I assembly, mitochondrial electron transport-NADH

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to ubiquinone and protein targeting to mitochondrion. Accordingly, gene set enrichment 9



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analysis showed that mitochondrial respiratory chain complex assembly (NES = 1.79 , p =

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0.000, q = 0.002 ), mitochondrial respiratory chain complex I biogenesis (NES = 1.78 , p =

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0.000, q = 0.003 ), mitochondrial translation (NES = 1.76 , p = 0.000, q = 0.004 ), and protein

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targeting to mitochondria (NES = 1.62 , p = 0.002, q = 0.045 ), were significantly enriched in

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the GA group (Fig. 1C). These data suggest that GA may regulate the mitochondrial function.

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GA induced mitochondrial fragmentation and reduced mitochondrial mass

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To verify the role of GA in mitochondria, we first examined the morphology of the

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mitochondria after GA treatment. The immunofluorescent staining of the mitochondrial

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marker HSP60 showed that GA dramatically induced mitochondrial fragmentation compared

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to the control group (Fig. 2A). Moreover, we found that GA decreased the expression of a

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mitochondrion outer membrane protein (TOM20) and cytochrome c oxidase polypeptide II

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(COX2), as well as decreased SUMO1- and SUMO2-modied proteins (Fig. 2B and S. Fig. 1).

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In addition, we detected significantly decreased relative mtDNA copy numbers in GA-treated

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HeLa cell compared with the DMSO treatment (Fig. 2C). These data indicated that GA

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reduced both the morphology and the mass of mitochondria.

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GA impaired mitochondria function

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To obtain insight into the difference in the mitochondrial functional between the GA- and

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DMSO-treated cells, we first mapped mitochondrial function by quantifying the oxygen

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consumption rate (OCR) using Seahorse® XF extracellular flux analyzer technology. The

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basal OCR showed no difference between the GA and DMSO treatments. Blockage of ATP

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synthase with oligomycin resulted in a decrease in the OCR to 60% of baseline under DMSO 10


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treatment but no changes under GA treatment. Addition of the mitochondrial membrane

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uncoupler FCCP resulted in an increased OCR in the control group but an unexpectedly

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decreased OCR in the GA group (Fig. 3A). The individual parameter calculations showed

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significantly lower levels of ATP-linked, maximal respiration and spare capacity, but

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dramatically higher proton leakage in GA-treated cells compared to DMSO treated (Fig. 3B).

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In addition, we measured the ATP levels of GA-treated and control cells using an ATP assay

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kit. Compared to the control group, GA treatment significantly decreased the ATP levels

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generated by the mitochondrial oxidative phosphorylation system after complete inhibition of

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glycolysis (Fig. 3C). These results suggested that GA impairs mitochondrial function.

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GA decreased mitochondrial biogenesis

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Next, we asked whether the reduced mitochondrial mass was caused by the suppression of

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mitochondrial biogenesis. As shown in Fig. 4A, GA treatment significantly decreased the

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mRNA levels of mitochondrial fission (FIS1) and fusion proteins (MFN1, MFN2),

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mitochondrial membrane proteins (TOM20, TIM23), mitochondrial electron transport chain

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proteins (SDHA, NDUFA6, NDUFB2) and molecular chaperones associated with

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mitochondrial function (HSPA1A, HSPA1B). Moreover, we examined the expression levels

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of PGC-1, the key transcription factor invovled in mitochondrial biogenesis in response to

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changes in the cellular environment or the physiological or pathological status of mammals33.

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We found that GA treatment dramatically decreased both the mRNA and protein expression

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of PGC-1 (Fig. 4B-C). Next, we treated the HeLa cells with PGC-1 agonist (ZLN005) or

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inhibitor (SR-18292) and examined the GA effect on mitochondrial proteins. We found that 11



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further treatment with PGC-1 agonist (ZLN005) could partially restore GA-mediated

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mitochondrial proteins loss (TIMM23 and SDHA) (Fig. 4D-F). Furthermore, we treated the

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cells with PGC-1 inhibitor (SR18292) for 24h and then with GA. As shown in Fig. 4G-H,

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SR18292 treatment decreased mitochondrial proteins, and further treatment with GA still

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decreased mitochondrial proteins. Together, these data suggested that the GA treatment

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reduced mitochondrial mass partially by blocking PGC-1-mediated mitochondria biogenesis.

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GA induced mitophagy

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Another possible reason for the decrease in mitochondrial mass is an increase in

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mitochondrial elimination, and mitophagy is a major way to eliminate damaged or no-longer-

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needed mitochondria. We detected a significant increase in LC3B-II lipidation and in the

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number of LC3 puncta (an indicator of autophagosome formation) from the GA-treated cells

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(S. Fig. 2), suggesting that GA activates autophagy. Next, the colocalization of mitochondria

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(green cytochrome c signal) and lysosomes (red LAMP1 signal, which can mark the ongoing

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autophagolysis of mitochondria), was examined by confocal microscopy. As shown in Fig.

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5A, C-D, the green and red fluorescence signals overlapped in most GA-treated cells.

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Similarly, increased colocalization of mitochondria and autophagosomes (red LC3 puncta)

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was found upon GA treatment (Fig. 5B-D). Furthermore, we detected a significanly reduced

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mitochondrial membrane potential, as indicated by increased JC1 green fluorescence under

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GA treatment, while most of the DMSO-treated cells exhibited JC1 red staining (Fig. 5E-F).

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To confirm that GA activates mitophagy, we asked whether autophagy inhibition would

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rescue the GA-induced mitochondrial mass loss. ATG7 knockout significantly rescued the 12


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GA-induced decrease in the expression of mitochondrial proteins including TOM20, TIM23,

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and SDHA, as well as induced increase in LC3B-II lipidation (Fig. 6A-B, and S. Fig. 3).

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Similarly, the downregulation effect of GA on mitochondrial TOM20 and COX2 was

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abolished by chloroquine (CQ) -mediated autophagy repression (Fig. 6C). Moreover, ATG7

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knockout abolished GA-induced mitochondrial fragmentation and reduced the relative

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mtDNA copy numbers (Fig. 6D-E). Taken together, these data demonstrated that GA induces

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mitophagy.

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GA induced FUNDC1-mediated mitophagy

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Next, we investigated which signaling pathway mediates GA induced mitophagy. PINK-

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Parkin-, BNIP3-, and FUNDC1- pathways are three common ones to mediate mitophagy. As

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HeLa cell lack endogenous Parkin

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found that GA treatment significantly decreased the mitochondrial proteins (TIM23, TOM20

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and SDHA) in both wild-type and Parkin-positive cells (S. Fig. 4), suggesting that GA

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induced a PINK-Parkin-independent type of mitophagy. Therefore, we evaluated GA function

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in BNIP3- and FUNDC1-knockdown HeLa cells FUNDC1 knockdown abolished the GA-

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induced repression of mitochondrial proteins (TOM20, SDHA, and TIM23) compared to the

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control cells (pLKO.1 group), but BNIP3 knockdown did not (Fig. 7A-B). Moreover, a GA-

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induced mtDNA reduction appeared in the control and BNIP3-knockdown cells, whicht

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disappeared in FUNDC1-knockdown cells (Fig. 7C). Furthermore, knockdown of FUNDC1,

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but not BNIP3, partially restored the MMP of GA-treated cells (Fig. 7D-E). In addition,

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FUNDC1 knockdown restored the reduced mRNA levels of some mitochondrial proteins,

34,

we ectopically expressed Parkin in HeLa cells.We

13



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including MFN2, TOM20, SDHA, HSPA1A, and NDUFB2, upon GA treatment (S. Fig. 5 and

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Fig. 4A). Furthermore, GA was shown to have activity as a sumoylation or HAT inhibitor 26,

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so we tested whether FUNDC1 can be regulated by sumoylation or acetylation. We

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performed sumoylation or acetylation site prediction by using the GPS website of the

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CUCKOO Workgroup (http://www.biocuckoo.org/) and found that there are no conserved

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sumoylation sites in FUNDC1, but the presence of a sumoylation interaction motif and two

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lysine acetylation sites (lysines 114 and 115) (S. Fig. 6) These data indicated that the GA-

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induced mitochondrial mass loss is also due to increased mitophagy, and that FUNDC1 is

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required for GA-induced mitophagy.

274 275

Discussion

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Previously, we revealed that GA (15:1) promoted adipocyte commitment but suppressed

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adipocyte terminal differentiation in mouse bone marrow stromal cells, possibly through its

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activity as a sumoylation inhibitor, but not its activity as a HAT inhibitor29. In addition, we

279

observed the elevated expression of mitochondrial genes in GA treated mBMSCs29. In the

280

present study, we investigated the role of GA in regulating mitochondrial morphology and

281

function. We found that GA treatment induced mitochondrial fragmentation, reduced mtDNA

282

copy numbers and mitochondrial proteins, and reduced ATP levels and OCRs (Fig. 2-3). In

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addition, GA treatment significantly reduced the mitochondrial membrane potential (Fig. 5E-

284

F), which is consistent with a previous study performed in MDCK and HepG2 cells in which

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GA treatment resulted in loss of the cell mitochondrial membrane potential and cell cycle

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arrest, which may contribute to cell death

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treatment signaficantly decreased the cell viabliliy (S. Fig. 7) GA has been used as a new

288

molluscicide agent for its significant mitochondrial damage and decreases the expression of

289

mitochondrial enzymes in snails 35.

290

Mitochondria are highly dynamic organelles that undergo constant fusion and fission, and

291

their mass may be balanced by mitochondrial biogenesis and mitophagy36. Mitochondrial

292

fusion and fission are both mediated by the large GTPases of the dynamin family, which are

293

conserved in different species 37. Mitofusins (Mfn1 and Mfn2) mediate mitochondrial outer

294

membrane fusion, while Optic atrophy 1 (OPA1) mediate the inner membrane fusion in

295

mammals

296

Dynamin related protein1 (Drp1), which is most well studied

297

mitochondrial outer membrane via a collection of receptor proteins (Mff, Fis1, MiD49, and

298

MiD50), and then it assembles around the tubule and constricts mitochondria in a GTP-

299

dependent manner to mediate scission

300

modifications, including phosphorylation, ubiquitination, S-nitrosylation and sumoylation 37.

301

MAPL Sumoylation of Drp1 stimulates mitochondrial fission and stabilizes an

302

ER/mitochondrial platform required for cell death

303

deSUMOylation of Drp1 increasing its association with mitochondria, cytochorome c release,

304

and facilitates interaction with Mff to promote cell death

305

treatment significantly decreased the mRNA levels of mitochondrial fission (Fis1) and fusion

38.

17, 27.

Consistently, we also detected that GA

Mitochondrial fission is mediated by several proteins, including GTPase

40.

39.

Drp1 is recruited to the

Drp1 is regulated by multiple post-translational

41,

42.

However, SENP3-mediated

43 44.

15


Our data showed that GA


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proteins (Mfn1, Mfn2), but did not change Drp1 mRNA expression (Figure 4A). GA can

307

directly bind to and inhibit sumoylation E1 (SAE1/SAE2) enzyme activity 26, suggesting that

308

GA may also inhibit the sumoylation of Drp1 to increase its association with mitochondria.

309

We found that GA-induced decrease in mitochondrial mass may be caused by decreased

310

mitochondrial biogenesis by peroxisome proliferator-activated receptor γ-coactivator-1α

311

(PGC-1) (Fig. 4B-C). PGC-1 is a major regulator of mitochondrial biogenesis in response

312

to changes in the cellular environment or the physiological or pathological status of

313

mammals45. It is regulated by many posttranslational modifications, including acetylation,

314

phosphorylation, methylation and sumoylation

315

been reported to acetylate and inhibit PGC-1 activity in vitro and in vivo

316

Sirt1-mediated deacetylation of PGC-1 leads to its activation

317

lysine 183 located in the activation domain of PGC-1 does not have an apparent effect on

318

the subcellular localization or stability of PGC-1α but does attenuate the transcriptional

319

activity of the coactivator

320

sumoylation E1 enzyme (SAE1/SAE2)

321

acetylation 26, 52. Our data showed that GA reduced both mRNA and protein levels of PGC-

322

1, so GA may regulate the upstream of PGC-1 through its sumoylation inhibitor or PCAF

323

inhibitor activity.

324

Moreover, we detected significant increases in LC3B-II lipidation and the number of LC3

325

puncta in GA-treated cells, (S. Fig. 2). Additionally, the combination of GA and CQ

326

(lysosomal inhibitors) further increased LC3B-II compared to GA treatment alone (Fig. 6C),

50, 51.

45.

The histone acetyltransferase GCN5 has

48, 49.

46, 47,

whereas

SUMO1 conjugated to

GA has been reported to directly bind to and inhibit the 26,

but it also inhibits PCAF mediated histone

16


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suggesting that GA increased the autophagy flux. Similarly, GA has been reported to suppress

328

colon cancer cell proliferation, migration, and invasion, and to inhibit the EMT in lung cancer

329

cells through repression of the mTOR signaling pathway, which is the major negative

330

regulator of autophagy20,

331

mitochondria and autophagosomes/ lysosomes. Additionally, autophagy inhibition induced

332

chemically or genetically can restore the GA-induced mitochondrial mass loss. These overall

333

demonstrate the function of GA in inducing mitophagy.

334

Since HeLa cells lack expression of Parkin, and GA treatment significantly decreased the

335

mitochondrial proteins (TIM23, TOM20 and SDHA) in both wild-type and Parkin-positive

336

cells (S. Fig. 4), thus the GA induced mitophage should act in an Parkin-independent manner.

337

It was shown that FUNDC1 knockdown, but not BNIP3 knockdown, blocked the activity of

338

GA in inducing mitochondrial fragmentation, and reducing mtDNA copy numbers and

339

mitochondrial protein levels (Fig. 7A-C), suggesting that GA induced FUNDC1-dependent

340

mitophagy. FUNDC1, a mitochondrial outer-membrane protein, is a hypoxia-induced

341

mitophagy receptor that interacts with and recruits LC3 to mitochondria for mitophagy

342

FUNDC1 is phosphorylated at tyrosine 18 (Y18) and serine 13 (S13) by SRC kinase and CK2,

343

reducing its affinity for LC3 13. Under hypoxic conditions, FUNDC1 is dephosphorylated by

344

PGAM5 or other yet-to-be-identified phosphatases, which greatly increases its interaction

345

with LC3 or other autophagy genes for the initiation of mitophagy 54. Thus, we also examined

346

the effects of GA on regulating SRC kinase, CK2 and PGAM5. We found that GA treatment

347

decreased the mRNA levels of SRC and CK2, but not PGAM5 (S. Fig. 8A). Moreover, GA

53.

Furthermore, we observed increased colocalization of

17


13.


348

treatment signalificant decreased the protein level of SRC and CK2 (S. Fig. 8B-C). These data

349

suggested that GA may decrease the phosphorylation of FUNDC1 to promote its interaction

350

with LC3, leading the initiation of mitophagy.

351

GA has been shown to inhibit both the sumoylation E1 enzymes and histone acetyltransferase,

352

which suggests that FUNDC1 may also be regulated by sumoylation and histone

353

acetyltransferase. The prediction results showed that there are no conserved sumoylation sites

354

in FUNDC1, but the presence of a sumoylation interaction motif (S. Fig. 6A) indicated that

355

FUNDC1 might interact with sumoylated proteins to mediate GA-induced FUNDC1-

356

dependent mitophagy. In addition, we predicted that lysines 114 and 115 of FUNDC1 may be

357

acetylated; in particular, lysine 115 of FUNDC1 may be acetylated by the histone

358

acetyltransferase KAT2A (GCN5) or KAT2B (PCAF) (S. Fig. 6B). Since GA inhibits PCAF-

359

mediated histone acetylation, we hypothesize that GA may block PCAF-mediated FUNDC1

360

acetylation, which results in mitophagy. In the future, we will peruse these hypotheses and

361

reveal the regulatory mechanism of FUNDC1, which may promote the understanding of the

362

exact role of FUNDC1 in mitophagy.

363

In conclusion, we found that GA treatment caused mitochondrial fragmentation reduced the

364

MMP and mtDNA levels, and repressed mitochondrial OCRs. Mechanistic studies further

365

revealed that GA-induced mitochondrial mass loss may result from decreased mitochondrial

366

biogenesis and increased FUNDC1-dependent mitophagy. Our work may provide new

367

mechanistic insights of GA in its future clinical applications for cancers or other diseases, as

368

well as the side effects of the ginkgo leaf extract for cosmetics and ginkgo nuts for food. 18


Page 18 of 39

Page 19 of 39


369

Abbreviations

370

GA: Ginkgolic acids

371

FUNDC1: FUN14 domain-containing protein 1

372

BNIP3: BCL2/adenovirus E1B 19 kDa protein-interacting protein 3

373

CQ: Chloroquine

374

LC3B: Microtubule-associated proteins 1A/1B light chain 3B

375

LAPM1: Lysosome-associated membrane glycoprotein 1

376

OCR: Oxygen consumption rate

377

FCCP: Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone

378

MMP: Mitochondrial membrane potential

379

HSP60: 60 kDa heat shock protein, mitochondrial

380

COX2: Ccytochrome c oxidase polypeptide II

381

FIS1: Mitochondrial fission 1 protein

382

MFN1: Mitofusin-1

383

MFN2: Mitofusin-2

384

TOM20: Mitochondrial import receptor subunit TOM20 homolog

385

TIM23: Mitochondrial import inner membrane translocase subunit Tim23

386

SDHA: Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial

387

NDUFA6: NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6

388

NDUFB2: NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 2, mitochondrial

389

HSPA1A: Heat shock 70 kDa protein 1A 19



390

HSPA1B: Heat shock 70 kDa protein 1B

391

PGC-1: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha

392

JC1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbo-cyanine iodide

393

Cyto c: Cytochrome c

394

mTOR: Mechanistic target of rapamycin

395

Acknowledgements

396

We would like to thank Dr. Quan Chen (Nankai University, China) and Dr. Zhiyin Song

397

(Wuhan University, China) for the ATG7 KO and control HeLa cells, and thank Dr. Xuhui

398

Lai for technical help on our qPCR procedure.

399

Funding

400

This work was financially supported by the National Natural Science Foundation of China

401

(Grants. 81601299, 81800833 and 81802189), the 111 Project (B16021), the National Key

402

R&D Program of China (2018YFC2002000), the China Postdoctoral Science Foundation

403

(Grant 2018M631054), and the Natural Science Foundation of Guangdong Province (Grant

404

2018A0303131002)

405

Competing interests

406

The authors declare no competing financial interest.

407

Authors’ contributions

408

Experiments were designed by J.S.L and Q.H.Z. Experiments were performed by W.J.W,

409

M.M.W and J.Y.T, Data analyzed by W.J.W, M.M.W, Y.R, T.Y and H.W provided technical 20


Page 20 of 39

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410

support and disscussions.W.J.W, J.S.L and Q.H.Z wrote the manuscript.. All the authors

411

reviewed the manuscript.

412 413

References

414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447

1. Jahani-Asl, A.; Germain, M.; Slack, R. S., Mitochondria: joining forces to thwart cell death. Biochimica et biophysica acta 2010, 1802, 162-6. 2. Williams, J. A.; Ding, W. X., Mechanisms, pathophysiological roles and methods for analyzing mitophagy - recent insights. Biological chemistry 2018, 399, 147-178. 3. Zhou, Q.; Li, H.; Li, H.; Nakagawa, A.; Lin, J. L.; Lee, E. S.; Harry, B. L.; SkeenGaar, R. R.; Suehiro, Y.; William, D.; Mitani, S.; Yuan, H. S.; Kang, B. H.; Xue, D., Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization. Science 2016, 353, 394-9. 4. Politi, Y.; Gal, L.; Kalifa, Y.; Ravid, L.; Elazar, Z.; Arama, E., Paternal mitochondrial destruction after fertilization is mediated by a common endocytic and autophagic pathway in Drosophila. Developmental cell 2014, 29, 305-20. 5. Gustafsson, A. B.; Dorn, G. W., 2nd, Evolving and Expanding the Roles of Mitophagy as a Homeostatic and Pathogenic Process. Physiological reviews 2019, 99, 853892. 6. Ryu, D.; Mouchiroud, L.; Andreux, P. A.; Katsyuba, E.; Moullan, N.; Nicolet-DitFelix, A. A.; Williams, E. G.; Jha, P.; Lo Sasso, G.; Huzard, D.; Aebischer, P.; Sandi, C.; Rinsch, C.; Auwerx, J., Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents. Nature medicine 2016, 22, 879-88. 7. Kim, M. J.; Yoon, J. H.; Ryu, J. H., Mitophagy: a balance regulator of NLRP3 inflammasome activation. BMB reports 2016, 49, 529-535. 8. Pavlides, S.; Vera, I.; Gandara, R.; Sneddon, S.; Pestell, R. G.; Mercier, I.; MartinezOutschoorn, U. E.; Whitaker-Menezes, D.; Howell, A.; Sotgia, F.; Lisanti, M. P., Warburg meets autophagy: cancer-associated fibroblasts accelerate tumor growth and metastasis via oxidative stress, mitophagy, and aerobic glycolysis. Antioxidants & redox signaling 2012, 16, 1264-84. 9. Bingol, B.; Sheng, M., Mechanisms of mitophagy: PINK1, Parkin, USP30 and beyond. Free radical biology & medicine 2016, 100, 210-222. 10. Sandoval, H.; Thiagarajan, P.; Dasgupta, S. K.; Schumacher, A.; Prchal, J. T.; Chen, M.; Wang, J., Essential role for Nix in autophagic maturation of erythroid cells. Nature 2008, 454, 232-5. 11. Zhang, T.; Xue, L.; Li, L.; Tang, C.; Wan, Z.; Wang, R.; Tan, J.; Tan, Y.; Han, H.; Tian, R.; Billiar, T. R.; Tao, W. A.; Zhang, Z., BNIP3 Protein Suppresses PINK1 Kinase Proteolytic Cleavage to Promote Mitophagy. The Journal of biological chemistry 2016, 291, 21616-21629. 21



448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488

12. Chen, M.; Chen, Z.; Wang, Y.; Tan, Z.; Zhu, C.; Li, Y.; Han, Z.; Chen, L.; Gao, R.; Liu, L.; Chen, Q., Mitophagy receptor FUNDC1 regulates mitochondrial dynamics and mitophagy. Autophagy 2016, 12, 689-702. 13. Liu, L.; Feng, D.; Chen, G.; Chen, M.; Zheng, Q.; Song, P.; Ma, Q.; Zhu, C.; Wang, R.; Qi, W.; Huang, L.; Xue, P.; Li, B.; Wang, X.; Jin, H.; Wang, J.; Yang, F.; Liu, P.; Zhu, Y.; Sui, S.; Chen, Q., Mitochondrial outer-membrane protein FUNDC1 mediates hypoxiainduced mitophagy in mammalian cells. Nature cell biology 2012, 14, 177-85. 14. Jacobs, B. P.; Browner, W. S., Ginkgo biloba: a living fossil. The American journal of medicine 2000, 108, 341-2. 15. Parsad, D.; Pandhi, R.; Juneja, A., Effectiveness of oral Ginkgo biloba in treating limited, slowly spreading vitiligo. Clinical and experimental dermatology 2003, 28, 285-7. 16. Ginkgo nuts nutrition facts 17. Qi, Q. M.; Xue, Y. C.; Lv, J.; Sun, D.; Du, J. X.; Cai, S. Q.; Li, Y. H.; Gu, T. C.; Wang, M. B., Ginkgolic acids induce HepG2 cell death via a combination of apoptosis, autophagy and the mitochondrial pathway. Oncology letters 2018, 15, 6400-6408. 18. Hua, Z.; Wu, C.; Fan, G.; Tang, Z.; Cao, F., The antibacterial activity and mechanism of ginkgolic acid C15:1. BMC biotechnology 2017, 17, 5. 19. Lu, J. M.; Yan, S.; Jamaluddin, S.; Weakley, S. M.; Liang, Z.; Siwak, E. B.; Yao, Q.; Chen, C., Ginkgolic acid inhibits HIV protease activity and HIV infection in vitro. Medical science monitor : international medical journal of experimental and clinical research 2012, 18, BR293-298. 20. Baek, S. H.; Ko, J. H.; Lee, J. H.; Kim, C.; Lee, H.; Nam, D.; Lee, J.; Lee, S. G.; Yang, W. M.; Um, J. Y.; Sethi, G.; Ahn, K. S., Ginkgolic Acid Inhibits Invasion and Migration and TGF-beta-Induced EMT of Lung Cancer Cells Through PI3K/Akt/mTOR Inactivation. Journal of cellular physiology 2017, 232, 346-354. 21. Chhunchha, B.; Singh, P.; Singh, D. P.; Kubo, E., Ginkgolic Acid Rescues Lens Epithelial Cells from Injury Caused by Redox Regulated-Aberrant Sumoylation Signaling by Reviving Prdx6 and Sp1 Expression and Activities. International journal of molecular sciences 2018, 19. 22. Yao, Q. Q.; Li, L.; Xu, M. C.; Hu, H. H.; Zhou, H.; Yu, L. S.; Zeng, S., The metabolism and hepatotoxicity of ginkgolic acid (17 : 1) in vitro. Chinese journal of natural medicines 2018, 16, 829-837. 23. Yoon, S. Y.; Lee, J. H.; Kwon, S. J.; Kang, H. J.; Chung, S. J., Ginkgolic acid as a dual-targeting inhibitor for protein tyrosine phosphatases relevant to insulin resistance. Bioorganic chemistry 2018, 81, 264-269. 24. Liu, J.; Li, Y.; Yang, X.; Dong, Y.; Wu, J.; Chen, M., Effects of ginkgol C17:1 on cisplatin-induced autophagy and apoptosis in HepG2 cells. Oncology letters 2018, 15, 10211029. 25. Li, J.; Li, A.; Li, M.; Liu, Y.; Zhao, W.; Gao, D., Ginkgolic acid exerts an antiinflammatory effect in human umbilical vein endothelial cells induced by ox-LDL. Die Pharmazie 2018, 73, 408-412.

22


Page 22 of 39

Page 23 of 39

489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531


26. Fukuda, I.; Ito, A.; Hirai, G.; Nishimura, S.; Kawasaki, H.; Saitoh, H.; Kimura, K.; Sodeoka, M.; Yoshida, M., Ginkgolic acid inhibits protein SUMOylation by blocking formation of the E1-SUMO intermediate. Chemistry & biology 2009, 16, 133-40. 27. Yao, Q. Q.; Liu, Z. H.; Xu, M. C.; Hu, H. H.; Zhou, H.; Jiang, H. D.; Yu, L. S.; Zeng, S., Mechanism for ginkgolic acid (15 : 1)-induced MDCK cell necrosis: Mitochondria and lysosomes damages and cell cycle arrest. Chinese journal of natural medicines 2017, 15, 375383. 28. Hecker, H.; Johannisson, R.; Koch, E.; Siegers, C. P., In vitro evaluation of the cytotoxic potential of alkylphenols from Ginkgo biloba L. Toxicology 2002, 177, 167-77. 29. Liu, H.; Li, J.; Lu, D.; Li, J.; Liu, M.; He, Y.; Williams, B. O.; Li, J.; Yang, T., Ginkgolic acid, a sumoylation inhibitor, promotes adipocyte commitment but suppresses adipocyte terminal differentiation of mouse bone marrow stromal cells. Scientific reports 2018, 8, 2545. 30. Bolte, S.; Cordelieres, F. P., A guided tour into subcellular colocalization analysis in light microscopy. Journal of microscopy 2006, 224, 213-32. 31. Venegas, V.; Halberg, M. C., Measurement of mitochondrial DNA copy number. Methods in molecular biology 2012, 837, 327-35. 32. Wang, W.; Wang, Q.; Wan, D.; Sun, Y.; Wang, L.; Chen, H.; Liu, C.; Petersen, R. B.; Li, J.; Xue, W.; Zheng, L.; Huang, K., Histone HIST1H1C/H1.2 regulates autophagy in the development of diabetic retinopathy. Autophagy 2017, 13, 941-954. 33. Cannino, G.; Di Liegro, C. M.; Rinaldi, A. M., Nuclear-mitochondrial interaction. Mitochondrion 2007, 7, 359-66. 34. Villa, E.; Marchetti, S.; Ricci, J. E., No Parkin Zone: Mitophagy without Parkin. Trends in cell biology 2018, 28, 882-895. 35. Xingliang Li ; Feng'e Deng ; Xiumei Shan ; Jiahu Pan ; Peizhong Yu ; Mao, Z., Effects of the molluscicidal agent GA-C13:0, a natural occurring ginkgolic acid,on snail mitochondria. Pesticide Biochemistry and Physiology 2012, 103, 115-120. 36. Pickles, S.; Vigie, P.; Youle, R. J., Mitophagy and Quality Control Mechanisms in Mitochondrial Maintenance. Current biology : CB 2018, 28, R170-R185. 37. van der Bliek, A. M.; Shen, Q.; Kawajiri, S., Mechanisms of mitochondrial fission and fusion. Cold Spring Harbor perspectives in biology 2013, 5. 38. Song, Z.; Ghochani, M.; McCaffery, J. M.; Frey, T. G.; Chan, D. C., Mitofusins and OPA1 mediate sequential steps in mitochondrial membrane fusion. Molecular biology of the cell 2009, 20, 3525-32. 39. Chan, D. C., Fusion and fission: interlinked processes critical for mitochondrial health. Annual review of genetics 2012, 46, 265-87. 40. Mishra, P.; Chan, D. C., Metabolic regulation of mitochondrial dynamics. The Journal of cell biology 2016, 212, 379-87. 41. Braschi, E.; Zunino, R.; McBride, H. M., MAPL is a new mitochondrial SUMO E3 ligase that regulates mitochondrial fission. EMBO reports 2009, 10, 748-54. 42. Prudent, J.; Zunino, R.; Sugiura, A.; Mattie, S.; Shore, G. C.; McBride, H. M., MAPL SUMOylation of Drp1 Stabilizes an ER/Mitochondrial Platform Required for Cell Death. Molecular cell 2015, 59, 941-55. 23



532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565

43. Guo, C.; Hildick, K. L.; Luo, J.; Dearden, L.; Wilkinson, K. A.; Henley, J. M., SENP3-mediated deSUMOylation of dynamin-related protein 1 promotes cell death following ischaemia. The EMBO journal 2013, 32, 1514-28. 44. Guo, C.; Wilkinson, K. A.; Evans, A. J.; Rubin, P. P.; Henley, J. M., SENP3mediated deSUMOylation of Drp1 facilitates interaction with Mff to promote cell death. Scientific reports 2017, 7, 43811. 45. Fernandez-Marcos, P. J.; Auwerx, J., Regulation of PGC-1alpha, a nodal regulator of mitochondrial biogenesis. The American journal of clinical nutrition 2011, 93, 884S-90. 46. Lerin, C.; Rodgers, J. T.; Kalume, D. E.; Kim, S. H.; Pandey, A.; Puigserver, P., GCN5 acetyltransferase complex controls glucose metabolism through transcriptional repression of PGC-1alpha. Cell metabolism 2006, 3, 429-38. 47. Kelly, T. J.; Lerin, C.; Haas, W.; Gygi, S. P.; Puigserver, P., GCN5-mediated transcriptional control of the metabolic coactivator PGC-1beta through lysine acetylation. The Journal of biological chemistry 2009, 284, 19945-52. 48. Gerhart-Hines, Z.; Rodgers, J. T.; Bare, O.; Lerin, C.; Kim, S. H.; Mostoslavsky, R.; Alt, F. W.; Wu, Z.; Puigserver, P., Metabolic control of muscle mitochondrial function and fatty acid oxidation through SIRT1/PGC-1alpha. The EMBO journal 2007, 26, 1913-23. 49. Canto, C.; Gerhart-Hines, Z.; Feige, J. N.; Lagouge, M.; Noriega, L.; Milne, J. C.; Elliott, P. J.; Puigserver, P.; Auwerx, J., AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature 2009, 458, 1056-60. 50. Rytinki, M. M.; Palvimo, J. J., SUMOylation attenuates the function of PGC-1alpha. The Journal of biological chemistry 2009, 284, 26184-93. 51. Cai, R.; Yu, T.; Huang, C.; Xia, X.; Liu, X.; Gu, J.; Xue, S.; Yeh, E. T.; Cheng, J., SUMO-specific protease 1 regulates mitochondrial biogenesis through PGC-1alpha. The Journal of biological chemistry 2012, 287, 44464-70. 52. Balasubramanyam, K.; Swaminathan, V.; Ranganathan, A.; Kundu, T. K., Small molecule modulators of histone acetyltransferase p300. The Journal of biological chemistry 2003, 278, 19134-40. 53. Liu, Y.; Yang, B.; Zhang, L.; Cong, X.; Liu, Z.; Hu, Y.; Zhang, J.; Hu, H., Ginkgolic acid induces interplay between apoptosis and autophagy regulated by ROS generation in colon cancer. Biochemical and biophysical research communications 2018, 498, 246-253. 54. Zhang, W.; Siraj, S.; Zhang, R.; Chen, Q., Mitophagy receptor FUNDC1 regulates mitochondrial homeostasis and protects the heart from I/R injury. Autophagy 2017, 13, 10801081.

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568 569

Fig. 1. GO and gene sets enrich analysis of gene expression profiling in GA- and DMSO-

570

treated HeLa cells. A. Heat map of diferential gene expression in GA- and DMSO-treated

571

HeLa cells (n = 2). B. The most up-regulated biologocal processes by DAVID GO analyses.

572

C. Gene Set Enrichment Analysis (GSEA) show the biological processes enriched in GA-

573

treated cells. (NES: normalized enrichment score; p: nominal p-value; q: false discovery rate

574

q-value).

575

25



576 577

Fig. 2. GA treatment induced mitochondrial fragmentation and reduced mitochondrial

578

mass.

579

A. Representative immunofluorescent staining of mitochondrial protein HSP60 following GA

580

treatment for 24 h in HeLa cells, quantificaion of the percentage of the cells with fully

581

fragmented mitochondria at the right (Scale bar = 10 μm, ** p < 0.01, **** p < 0.0001). B.

582

Wester blots of COX2 and TOM20 upon DMSO and GA treatment for 24h (50 M GA),

583

representative of 3 independent experiments. C. Measurement of mtNDA (mitochondrial

584

DNA) copy numbers in DMSO- and GA-treated HeLa cells by q-PCR (50 M GA for 24 h;

585

n=3, * p < 0.05 compared with the DMSO group).

586 587

26


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Page 27 of 39


588 589

Fig 3. GA repressed mitochondrial oxygen comsumption rate and ATP levels.

590

A. HeLa cell were seeded in the Seahorse Bioscience microplates (10,000 cells/well).

591

Following adherence for 12h, GA (50 M) or DMSO was added to the microplates for co-

592

incubation with cells for 12h, and 1M oligomycin, 0.5M FCCP and 0.5M

593

rotenoe/Antimycin A were subsequently added for mitochondrial OCR (oxgen comsuption

594

rate) measurement. B. Individual parameter for respiration, including basal respiration, proton

595

leak, ATP-linked oxygen consumption, maximal respiration and spare respiratio capacity in

596

HeLa cells ( n=3, ** p < 0.01; *** p < 0.001, compared with DMSO group) C. Mitochondria

597

oxidative phosphorylation system greated-ATP level measurement in HeLa cells. After cell

598

adherence for 12h, the culture medium were changed to DMEM (no glucose, 10% FBS)

599

medium, and GA (50M) or DMSO were added for 12h or 24h.The ATP levels were

600

measured by an Illuminometer, and normalized to the protein contents (n=3, * p < 0.05; ** p

601

< 0.01)

27



602 603

Fig. 4. GA repressed mitochondrial biogenesis. A. qPCR analysis the mRNA expression

604

levels of mitochondria related genes, including mitochondrial fission and fusion proteins,

605

mitochondrial membrane proteins, mitochondrial electron transport chain proteins and

606

molecular chaperones associated with mitochondrial function (n=3,* p < 0.05, compared to

607

DMSO treatment, GA: 50M for 24 h) . B. qPCR analysis of transcripiton factors to

608

mitochondrial biogenesis (n=3,* p < 0.05, compared to DMSO treatment, GA: 50M for 24

609

h). C. Representative western blots of PGC1a upon GA or DMSO treatment, quantification at 28


Page 28 of 39

Page 29 of 39


610

the right. (n=3,* p < 0.05; GA: 50M for 24 h). D. mRNA level of PGC1A in HeLa cells

611

upon ZLN005 treatment (ZLN005 for 24 h , n=3,**p < 0.01). E-F. Representative western

612

blots (E) and quantification (F) of mitochondrial proteins upon GA and ZLN005 treatment in

613

HeLa cells (GA: 50 M for 24 h; ZLN005: 10 M for 24 h; * p < 0.05; **p < 0.01). G-H.

614

Representative western blots (G) and quantification (H) of mitochondrial proteins upon GA

615

and SR18292 treatment (GA: 50 M for 24 h; SR18292: 20 M for 24 h; * p < 0.05; **p

Ginkgolic acids impairs mitochondrial function by decreasing

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