Ultraviolet-Ray-Induced Sea Cucumber (Stichopus japonicus) Melting

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Ultraviolet rays induced sea cucumber (Stichopus japonicus) melting is mediated through the caspase-dependent mitochondrial apoptotic pathway Li Su, Jing-Feng Yang, Xi Fu, Liang Dong, Da-Yong Zhou, Li-Ming Sun, and Zhenwei Gong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03888 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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Journal of Agricultural and Food Chemistry

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Ultraviolet rays induced sea cucumber (Stichopus japonicus) melting is mediated

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through the caspase-dependent mitochondrial apoptotic pathway

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Li Su†, Jing-Feng Yang†*, Xi Fu†, Liang Dong†, Da-Yong Zhou†, Li-Ming Sun†,

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Zhenwei Gong‡*

6 †

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School of Food Science and Technology, Dalian Polytechnic University, National

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Engineering Research Center of Seafood, No.1 Qinggongyuan, Ganjingzi district,

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Dalian 116034, P. R. China

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‡

Division of Pediatric Endocrinology, Department of Pediatrics, Children's

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Hospital of Pittsburgh of the University of Pittsburgh Medical Center, University of

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Pittsburgh School of Medicine, 4401 Penn Ave, Pittsburgh, Pennsylvania 15224,

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USA

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*Corresponding authors: Dr. Jing-Feng Yang

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E-mail address: [email protected], Tel: +86-411-86318729

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and

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Dr. Zhenwei Gong

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E-mail address: [email protected], Tel: +1-412-692-9601

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ACS Paragon Plus Environment


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Abstract

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Sea cucumber body wall melting occurs under certain circumstances. We have

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shown that apoptosis, but not autolysis, plays a critical role in the initial stage.

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However, it is still unclear how apoptosis is triggered in this process. In this study, we

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examined the reactive oxygen species (ROS), levels of B-cell lymphoma-2 (Bcl-2)

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and Bcl-2 Associated X Protein (Bax) and the depolarization of mitochondria

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transmembrane potential and Cytochrome c (Cyt c) release during the sea cucumber

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melting induced by ultraviolet (UV) exposure. We also investigated the contribution

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of caspase in this process by injecting pan-caspase inhibitor. Our data showed that UV

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exposure stimulates the ROS production, dysfunction of mitochondria and the release

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of Cyt c in sea cucumber coelomic fluid cells and body wall. We found a decrease of

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Bcl-2 and increase of Bax in the mitochondria after UV exposure. We also

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demonstrated that these changes are associated with the elevated activity of caspase-9

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and -3. Finally, our data showed that inhibition of caspase -9 and -3 using inhibitor

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suppresses the UV induced sea cucumber melting. These results suggest that the

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apoptosis during sea cucumber melting is mediated through the mitochondria

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dysfunction and following activation of caspase signaling pathway. This study

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presents a novel insight into the mechanism of sea cucumber melting.

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Key words: Stichopus japonicus, melting, apoptosis, autolysis, mitochondria

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Introduction

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Sea cucumber (Stichopus japonicus) is a well-known aquatic product in China and

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its production reached more than 200,000 tons in 2015 with an industrial output value

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exceeding 6 billion US dollars 1. In severe cases, such as expose to high temperature,

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salt concentration, nutrient deficiency and exposure to sunlight or UV irradiation, the

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sea cucumber body melting may occur, leading to the death of the animals 2. This

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phenomenon is a major concern for sea cucumber production, particularly during

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cultivation, transportation, handling and processing 3. Thus, understanding the sea

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cucumber melting mechanism could contribute to the sea cucumber cultivation and

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processing industry.

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The sea cucumber melting has long been recognized as a mechanism of autolysis4-6,

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which is commonly known as self-digestion of cells through the actions of their own

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enzymes. Our previous study has demonstrated that the initiation of the sea cucumber

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melting triggered by UV exposure is attributing to apoptosis but not autolysis 7. But

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the mechanism by which apoptosis is triggered remains unknown.

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Apoptosis can be triggered through two main pathways: the extrinsic (death

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receptor-dependent) and intrinsic (mitochondrial-dependent) pathways 8. The intrinsic

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pathway is a response to endogenous and exogenous factors, which causes the

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mitochondrial membrane becoming permeable and depolarized. Subsequently, the Cyt

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c is released from mitochondria to the cytosol leading to the elevation of cytosolic Cyt

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c level 9. The cytosolic Cyt c then binds to apoptotic protease activating factor 1

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followed by the activation of caspase

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. Cytosolic Cyt c is a key component and 3



11

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indicator of an apoptotic event

. Mitochondrial membrane permeability, i.e.

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depolarization of mitochondrial membrane potential, has also been proposed to play a

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role in the mitochondrial apoptotic cascade 12.

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In the current study, we sought to further investigate the molecular mechanism of

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apoptosis in sea cucumber melting phenomenon and reveal its signaling pathway. We

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examined the Cyt c release from mitochondria and the change of mitochondrial

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transmembrane potential in sea cucumber coelomic fluid cells after UV exposure. In

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addition, we investigated the changes in levels of ROS, Bcl-2 and Bax and activity of

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caspase-9 and -3 during sea cucumber melting after UV exposure.

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Materials and Methods Materials and Chemicals

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Sexually mature adult sea cucumbers (S. japonicus, 100-120 g; 15-20 cm total body

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length) were obtained from Dalian Liujiaqiao Seafood Market. Antibodies used for

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western blotting experiments are listed as following. Bax rabbit monoclonal antibody

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(Rabbit Antibody, react with Mouse, Rat, Human, Chinese Hamster and predicting

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react with Cow), anti-COX IV rabbit polyclonal, β-actin rabbit polyclonal antibody

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and a secondary anti-rabbit IgG (HRP-linked antibody) were purchased from Cell

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Signaling Technology (Beverly, MA, USA). The Bax antibody was generated using

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synthetic Human Bax peptide aa 1-100 (N terminal) as antigens. Anti-Bcl-2 rabbit

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polyclonal antibody (Rabbit Antibody, react with Human and predicting react with

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Mouse, Rat, Chicken, Cow, Cat and Dog) was bought from Abcam (Abcam,

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Cambridge, Mass, USA). The Bcl-2 antibody was generated using synthetic peptide 4


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within Human Bcl-2 aa 1-239 as antigen. The Enhanced BCA Protein Assay Kit was

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obtained from Beyotime Institute of Biotechnology (Shanghai, China). Sodium

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dodecyl sulphate (SDS), N,N,N,N-tetramethylethylenediamine (TEMED), Coomassie

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Brilliant Blue R-250, Bovine serum albumin (BSA) and the Tissue or Cell Total

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Protein Extraction Kit were from Sangon Biotech Co., Ltd. (Shanghai, China). The

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PVDF membrane was obtained from Immobilon (Millipore, Billerica, USA). All other

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chemicals used in this study were of analytical grade.

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Inducing sea cucumber melting by UV radiation

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Sea cucumber melting induced by UV was performed as previously described 7.

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Briefly, six groups of sea cucumber (seven samples per group) were exposed to UV

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light (wavelength 253.7nm, Cnlight, ZW20S19W, Jiangyin, Jiangsu, China) for half

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hour at intensity of 0.056 mw/cm2. After UV exposure, the sea cucumbers were

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transported to incubator with surroundings at 20 °C in dark (covered with Aluminum

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foil) and held for 0, 60, 120, 240 and 360 min respectively followed by apoptotic

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analysis. Untreated fresh sea cucumbers were used as control.

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Tissue preparation

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The UV-treated sea cucumbers were cut at the end of cloacal aperture. Midsection

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of sea cucumber body wall was sampled to tissue blocks (1 cm×1 cm×0.5 cm) and

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fixed in 10% neutral formalin in phosphate buffered saline (PBS, pH 7.4), embedded

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in paraffin and processed as previously described 7. Briefly, after fixation for 24 h,

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tissues were dehydrated, paraffin embedded, sectioned for histological experiments.

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Sections of 5 µm thick were heated in a distilled water bath (45 °C), collected on a 5



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glass slides, deparaffinised and rehydrated stepwise through an ethanol series and

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processed for routine histological analysis.

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Cyt c immunohistochemistry

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Sections from above were subjected to an antigen retrieval procedure by heating the

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samples at 65 °C for 30 min followed by cooling down for 10 min and then rinsing

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with dimethylbenzene, ethanol and distilled water each for 5 min. The sections were

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incubated in 3% hydrogen peroxide for 8 min to inactivate endogenous peroxidase in

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samples. Antigen retrieval was done by boiling sections in 0.01 M citrate (Sigma

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Aldrich, St. Louis, MO, USA, pH 6.0) and then rinsing with PBS for 5 min at room

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temperature followed by immersion in 5% bovine serum albumin (BSA, Sangon

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Biotech, Shanghai, China) at room temperature for 10 min. The processed sections

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were incubated with Cyt c antibody (BioLegend, San Diego, CA, USA) at 4°C

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overnight and were held in room temperature for 30 min followed by washing with

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PBS for 3 times. Sections were then incubated with goat anti-rabbit IgG-HRP (1/500

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in PBS, SE13, solarbio, Beijing, China) for 60 min at 37°C followed by PBS washing

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for 3 times. Sections were subsequently treated with 0.05% diaminobenzidine (DAB,

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Beyotime, Shanghai, China) for 10 min at room temperature, rinsed in PBS for 5 min

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and allowed to dry in the air.

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Mitochondrial membrane potential analysis

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The Jiamay BiotechTM JC-1 Assay Kit (Beijing, China) was used to quantify

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mitochondrial transmembrane potential. The cationic lipid fluorescent dye JC-1 was

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used to study depolarization of mitochondria transmembrane potential (decreasing red 6


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fluorescence density) and release of the JC-1 monomer into the cytoplasm (green

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fluorescence). Coelomic fluid cells (500 µL) extracted from treated samples were

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incubated with 500 µL of JC-1 (diluted 200 times according to the instructions) at 4°C

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for 10 minutes. The cells were washed with PBS and then analyzed using a flow

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cytometer (FACSVerse, Becton, Dickinson and Company, USA) at excitation 585 nm

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and emission 590 nm for the JC-1 aggregate (red) and at excitation 515 nm and

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emission 529 nm for the JC-1 monomer (green).

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ROS generating analysis

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DCFDA Cellular ROS Detection Assay Kit (Abcam, Sydney, Australia) was used

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for quantitative measurement of cellular ROS in sea cucumber coelomic fluid cells.

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After incubating with 5 µM DCFDA at room temperature for 30 min, coelomic fluid

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cells obtained from control and treated sea cucumbers were subjected to laser

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scanning confocal (SP8, Leica, Heidelberg, Germany) for ROS detection.

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Western Blotting analysis

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Western blotting analysis was done to determine the protein levels of Bax and Bcl-2

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in sea cucumber coelomic fluid cells after UV treatment. Cytoplasmic and

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mitochondrial proteins were extracted using cytoplasmic and mitochondrial protein

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extraction kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s

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instructions. Briefly, samples from different groups were homogenized in ice-cold

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cytoplasmic extraction buffer using glass pestle. After incubating for 15 min on ice,

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the pellets were collected and centrifuged at 900 g for 10 min at 4 °C. The

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supernatants were transferred to new tubes and then centrifuged at 13,800 g at 4 °C 7



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for 30 min to obtain the cytoplasmic protein in the supernatants. The pellets were

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re-suspended in cytoplasmic extraction buffer and centrifuged at 16,200 g at 4 °C for

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10 min. The pellets containing mitochondrial fraction were re-suspended in

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mitochondrial lysis buffer for 30 min on ice and then centrifuged at 16,200 g at 4 °C

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for 10 min to obtain the mitochondrial protein in the supernatants. Protein

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concentration was determined by the modified Bradford protein assay kit (Sangon

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Biotech, Shanghai, China).

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Protein samples (20 µg) from each group were resolved on 12% sodium dodecyl

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sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto

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polyvinyl difluoride (PVDF) membranes (Millipore, Billerica, America). The

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membranes were blocked with 5% non-fat milk in Tris Buffered Saline with 0.05%

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Tween 20 (TBS-T) and then incubated with primary antibodies (Bcl-2, 1:1,000, Bax,

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1: 10,000, β-Actin, 1:1,000, COX IV, 1:1,000) overnight at 4 °C. Membranes were

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washed three times with 1×TBS-T for 15 min, and exposed to secondary antibody

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(1:5,000) for 1 h at room temperature. Proteins were detected by Enhanced

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chemiluminescence (ECL) reagents (Nacalai Tesque, Kyoto, Japan).

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The influence of inhibitor on sea cucumber melting and the activity of Caspase-9 and

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Caspase-3

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The pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone

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(Z-VAD-FMK, KeyGEN Biotech, Nanjing, China) was dissolved in dimethyl

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sulfoxide (DMSO) and diluted to 20 µM with PBS. The inhibitor (2 mL) was injected

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to each sea cucumber from cloacal aperture and exposed to UV light and processed as 8


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indicated above. Caspase-9 and caspase-3 activities were determined using the

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Caspase-9 and Caspase-3 Colorimetric Assay Kit (KeyGEN Biotech, Nanjing, China)

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followed the manufacturer’s instruction. Briefly, sea cucumber intestine or coelomic

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fluid cells (0.3 g wet weight) were homogenized in ice-cold lysis buffer and

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centrifuged at 9,600 g for 5 min at 4°C to collect the supernatants for the assay. Fifty

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microliter of 2X Reaction Buffer (containing 10 mM DL-Dithiothreitol) was mixed

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with 50 µL of sample in a 96-well plate followed by addition of 5 µL of caspase-9 or

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caspase-3 substrate (50 µM final concentration) and 4 hours of incubation at 37 °C.

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The absorbance was read at 405 nm in a Microplate reader (Tecan, Männedorf,

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Switzerland) and the results were calculated as fold increases compared with

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untreated samples.

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Statistical Analysis

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All experiments were done at least 3 times independently with three replicates (n=3)

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for each time. Data are presented as mean ± standard deviation (SD). Mean values

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were compared by One-factor Analysis of Variance (ANOVA) and the differences

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between means were evaluated by using S-N-K test as well as T-test. The statistical

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analysis was performed by using SPSS 16.0 software (SPSS Inc. Chicago, IL, USA).

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Comparisons that yielded p values < 0.01 were considered significant.

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Results UV exposure induces Cyt c release from the mitochondria

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The distribution of Cyt c in the body wall during sea cucumber melting induced by

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UV was analyzed by the immunohistochemistry. We showed that the Cyt c in samples 9



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without UV exposure are uniformly dispersed in the body wall with clear edge (Figure

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1a). Nevertheless, the Cyt c in UV-exposed tissue samples had no clear edge (Figure

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1b-1f). These results demonstrated that the release of Cyt c from mitochondria to

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cytoplasm occurs within 0-6 hours after the UV exposure. The immunohistochemistry

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analysis was also performed using the inner body wall tissues and we found a similar

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pattern as in epidermis of the body wall tissues (Figure 2).

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UV irradiation causes depolarization of the mitochondrial transmembrane potential

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The change of mitochondrial membrane potential (∆Ψm) is a marker of 13

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mitochondrial function and is associated with apoptosis at early stage

. A cationic

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dye JC-1 was used to monitor the change of ∆Ψm in the sea cucumber coelomic fluid

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cells. Flow cytometry was used to measure the red/green fluorescence ratio (JC-1

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aggregate/JC-1 monomer ratio) as an indication of ∆Ψm. We showed that the ratios of

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red/green fluorescence intensity gradually decrease in the UV treated groups

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compared to the control group (0.1% DMSO) (Figure 3A and 3B). The ratios

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decreased from 5.05 to 3.97, 1.90, 1.65 and 1.69 in samples holding for 0h, 1h, 2h, 4h

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and 6 h post UV exposure. These observations suggest that the depolarization of ∆Ψm

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occurred in coelomic fluid cells of sea cucumber after UV treatment.

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UV exposure increases ROS production in sea cucumber coelomic fluid cells

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The ROS level in coelomic fluid cells after UV exposure were detected by DCFDA

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Cellular ROS Detection Kit and the ROS was labeled in green as shown in Figure 4A.

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The quantification data demonstrated that compared to the controls, UV exposure

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causes significantly increase in intracellular ROS and it continues to increase when 10


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hold in room temperature. We observed that the ROS level peaks at 2 hours after UV

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treatment and declines at 4 and 6 hours after UV treatment (Figure 4B). These results

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indicated that UV exposure leads to the increase in ROS levels in coelomic fluid cells

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at early time points.

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UV exposure induces Bax translocation

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The changes of Bcl-2 and Bax in both cytoplasmic and mitochondrial fractions of

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sea cucumber coelomic fluid cells were examined using western blotting. We

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demonstrated that the Bcl-2 is undetectable and Bax decreases in cytoplasmic fraction

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after the UV exposure (Figure 5A). We also found that the levels of Bax in

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mitochondrial fraction can hardly be detected at the earlier time points, but increased

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2 h after the UV exposure in a time dependent manner (Figure 5B). However, the

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Bcl-2 in mitochondrial fraction dropped (Figure 5C). We quantified the ratio of Bax to

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Bcl-2 in mitochondrial fraction and showed that the ratio of Bax/Bcl-2 in

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mitochondrial fraction increased significantly from 2 to 6 hour (Figure 5D).

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Pan-caspase inhibitor suppresses Caspase-3 and Caspase-9 activities induced by UV

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exposure

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After the UV treatment, the Caspase-3 activity increased significantly from 1 to

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2.94-fold in the intestinal cells and from 1 to 2.83-fold in coelomic fluid cells within 6

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hours (Figure 6A and 6B). Notably, the increase of caspase-3 activity induced by UV

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treatment was greatly inhibited by injection of Z-VAD-FMK, a pan-caspase inhibitor.

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A similar pattern was also observed in Caspase-9 activity both in coelomic fluid cells

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and intestinal cells (Figure 7). 11



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Caspase inhibitor suppresses sea cucumber body wall melting

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Z-VAD-FMK was injected into the fresh sea cucumbers from the cloacal aperture

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prior to the UV treatment. After 30 min UV exposure and 47 h left at 20 °C, the

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inhibitor treated sea cucumbers appeared identical to the fresh group sea cucumbers.

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However, the UV treated sea cucumbers without inhibitor injection showed melting

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phenomenon 20 h after UV treatment (Figure 8). This demonstrated that the

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pan-caspase inhibitor blocks the sea cucumber melting and the UV induced sea

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cucumber melting is mediated through the caspase-dependent pathway.

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Discussion

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Sea cucumbers initiate self melting upon environmental change, including changes

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in temperature, and salt concentration, and exposure to sunshine and UV. It was

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believed that autolysis plays key role in this process 4. However, our previous study

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showed that apoptosis, but not autolysis is the major cause contributing to the sea

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cucumber melting induced by UV exposure 7. We demonstrated that apoptosis appears

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in more than 50% of total cells in the body wall of sea cucumber within 6 h after UV

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exposure 7. The sea cucumber body wall is mainly composed of collagen with small

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amounts of cells. In our study, the apoptosis was analyzed in multiple tissues and cells

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such as the body wall, intestinal cells and body coelomic fluid cells. The evidence

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supports that the apoptosis appears not only in cell level but also in tissue level.

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Two major signaling pathways have been discovered to mediate the cellular

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apoptosis, the intrinsic and extrinsic pathways 14. Mitochondria are well known to be

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involved in the intrinsic pathway and play a critical role in apoptosis 12


15

. The

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mitochondrial pathway of cell apoptosis is regulated through caspase-9 16. It has been

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reported that the release of Cyt c causes the activation of caspase-9 through the

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apoptosome and then activates caspase-3 to induce cell death 17. The decrease in ∆Ψm

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and the release of Cyt c are two key steps for activating mitochondria mediated

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apoptosis 18. In the current study, UV exposure was used to trigger the sea cucumber

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melting and the results demonstrated that UV exposure induces the release of Cyt c

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and mitochondrial dysfunction in sea cucumbers (Figure 1, 2 and 3).

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The loss of ∆Ψm is a hallmark and early event of apoptosis coinciding with caspase 19

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activation

. Two hours after exposing to UV, sea cucumber coelomic fluid cells

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exhibited a collapse of ∆Ψm, which may further lead to the release of Cyt c from

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mitochondria into the cytosol. Indeed, we observed the increase of Cyt c release from

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mitochondria and the collapse of ∆Ψm in sea cucumber cells after exposed to UV,

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suggesting that both the Cyt c release and collapse of ∆Ψm play critical roles in UV

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induced apoptosis in sea cucumber.

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Cyt c works on the electron transport chain during respiration in the mitochondria.

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It transfers an electron from respiratory Complex III to Complex IV in the inner

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mitochondrial membrane

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release of Cyt c. The release of Cyt c from mitochondria into the cytoplasm triggers a

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cascade of reactions leading to apoptosis 18 ,21. The release of Cyt c from mitochondria

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is generally preceded by mitochondrial stress that starts with receiving an apoptotic

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signal from outside or internal factors

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cucumber mitochondria likely through the oxidative stress and the collapse of ∆Ψm. It

20

. The apoptotic signals affect the ∆Ψm and lead to the

22

. The UV exposure may cause stress to sea

13



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has been reported that sea cucumber tissue sample generates reactive oxygen species

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(ROS) and oxidative stress after UV exposure, which might be involved in sea

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cucumber melting 23. Our study also showed an increased ROS in coelomic fluid cells

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after the sea cucumber exposed to UV and held in room temperature (Figure 4). ROS

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induces cardiolipin peroxidation in the mitochondrial inner membrane, thus causing

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Cyt c release due to the breach of electrostatic/hydrophobic interactions 24.

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The Bcl-2 family members serve as either activators (e.g., Bax or Bad) or inhibitors 12

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(e.g., Bcl-2, Bcl-xL) for cellular apoptosis

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mitochondrial membrane, Cyt c release and activation of caspase-9 and caspase-3

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cascade 25. It is demonstrated that the overexpression of Bcl-2 prevents Cyt c release

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but Bax enhances it

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level of Bcl-2 in mitochondria (Figure 5C), and the level of Bax decreased in

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cytoplasm but increased in mitochondria after the UV exposure. These results

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demonstrated that the Bax spontaneously translocates to the mitochondria from

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cytoplasm 2 h after the UV exposure. Upon delivery of an apoptotic stimulus,

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cytoplasmic Bax translocates to the outer mitochondrial membrane, where it forms

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homodimers, creating pores that causes loss of ∆Ψm, Cyt c release from the

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intermembrane space of the mitochondrion into the cytosol 28. The reduction of Bcl-2

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in mitochondria and the translocation to the mitochondria of Bax in sea cucumber

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cells lead to the Cyt c release from the mitochondria into the cytoplasm and activation

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of caspase-9 and caspase-3.

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. They regulate the permeability of the

26,27

. We showed in this study that the UV exposure reduces the

The activation of the initiators of apoptosis, caspase-3 and -9, were observed in the 14


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UV treated sea cucumber cells (Figure 6 and 7). The caspase family proteins exist as

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inactive precursor form in the cytosol. Upon activation by apoptotic signals, the

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precursors of caspase are cleaved and processed to generate active enzymes through

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proteolytic pathways

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irreversibly binds to the catalytic site of caspase proteases and blocks up the apoptosis

314

19

. Z-VAD-FMK is a cell-permeable pan-caspase inhibitor that

29

. Our data demonstrated that the injection of inhibitor results in the suppression of

315

caspase-3 and caspase-9 activation in UV induced sea cucumber, indicating the

316

feasibility of applying Z-VAD-FMK to inhibit the activity of caspase-3 and -9 in vivo

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in sea cucumber. In addition, we showed that Z-VAD-FMK injection mostly blocks

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the UV induced sea cucumbers melting (Figure 8). Since activation of caspase

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cascade is a pro-apoptotic signal 21, and Z-VAD-FMK is an effective inhibitor to the

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caspase proteases, we conclude that both caspase-3 and -9 contribute to the apoptosis

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in sea cucumber cells induced by UV exposure and the sea cucumber melting is

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mediated through the caspase-dependent mitochondrial apoptotic pathway.

323

Taking together, our study demonstrated that the UV exposure increases the ROS

324

production, collapse of ∆Ψm and Cyt c release from the mitochondria into the cytosol,

325

therefore activating caspase-9 and caspase-3 dependent apoptosis in the sea cucumber

326

cells. Our study demonstrated that the apoptosis in sea cucumbers is mediated through

327

the reduction in Bcl-2 and induction Bax in the mitochondria after UV treatment. The

328

current studies further revealed that apoptosis plays critical roles in the sea cucumber

329

melting, and that UV induced apoptosis in sea cucumber is mediated through the

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caspase-dependent mitochondrial apoptotic pathway. 15



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Funding Sources

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This work is supported by “The National Natural Science Foundation” (No.

333

31771998，No. 31301430), “Natural Science Foundation of Liaoning Province” (No.

334

20170540062), Support of National Engineering Research Center of Seafood from

335

National

336

2012FU125X03, Third batch of Liaoning Collaborative Innovation Center from

337

provincial department of public education LJF[2015]NO.99.

Ministry

of

Science

and

Technology

338

The authors declare no competing financial interest.

339

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M.; Liu, Y.; Murata, Y. Purification and partial characterisation of a cathepsin

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L-like proteinase from sea cucumber (Stichopus japonicus) and its tissue

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distribution in body wall. Food Chem. 2014, 158, 192-199.

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6. Wilkie, I. Is muscle involved in the mechanical adaptability of echinoderm mutable collagenous tissue? J. Exp. Biol. 2002, 205, 159-165.

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7. Yang, J.; Gao, R.; Wu, H.; Li, P.; Hu, X.; Zhou, D.; Zhu, B.; Su, Y. Analysis of

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Apoptosis in Ultraviolet-Induced Sea Cucumber (Stichopus japonicus) Melting

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Using

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End-Labeling Assay and Cleaved Caspase-3 Immunohistochemistry. J. Agric.

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Food Chem. 2015, 63, 9601-9608.

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Terminal

Deoxynucleotidyl-Transferase-Mediated

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8. Martin, D.; Baehrecke, E. Caspases function in autophagic programmed cell death in Drosophila. Development. 2004, 131, 275-284.

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9. Sanz-Blasco, S.; Valero, RA.; Rodríguez-Crespo, I.; Villalobos, C.; Núñez, L.

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Mitochondrial Ca2+ overload underlies Abeta oligomers neurotoxicity providing

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an unexpected mechanism of neuroprotection by NSAIDs. PLoS One. 2008 7,

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10. Lee, J.; Park, Y.; Pun, S.; Lee, S.; Lo, J.; Lee, L. Real-time investigation of

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cytochrome c release profiles in living neuronal cells undergoing amyloid beta

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oligomer-induced apoptosis. Nanoscale. 2015, 7, 10340-10343.

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11. Hengartner , MO. The biochemistry of apoptosis. Nature. 2000 , 407, 770-776.

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12. Wu, Z.; Sun, H.; Li, J.; Ma, C.; Zhao, S.; Guo, Z.; Lin, Y.; Lin, Y.; Liu, L. A

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polysaccharide from Sanguisorbae radix induces caspase-dependent apoptosis in

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human leukemia HL-60 cells. Int. J. Biol. Macromol. 2014, 70, 615-620.

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13. Roy, S.; Hajnoczky, G. Calcium, mitochondria and apoptosis studied by fluorescence measurements. Methods. 2008, 46, 213-223.

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14. Green, D.; Evan, G. A matter of life and death. Cancer Cell. 2002, 1, 19-30.

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15. Adrain, C.; Martin, S. The mitochondrial apoptosome: a killer unleashed by the

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cytochrome seas. Trends Biochem. Sci. 2001, 26, 390-397.

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Processing of apoptosis-inducing factor (AIF). Biochem. Biophys. Res. Commun.

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2010, 396, 95-100.

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17. Nalepa, G.; Zukowska, E. Caspases and apoptosis: Die and let live. Wiad. Lek. 2002, 55, 100-106.

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18. Garrido, C.; Galluzzi, L.; Brunet, M.; Puig, P.; Didelot, C.; Kroemer, G.

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Mechanisms of cytochrome c release from mitochondria. Cell Death Differ. 2006,

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Huang, F. Strophalloside Induces Apoptosis of SGC-7901 Cells through the

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Mitochondrion-Dependent Caspase-3 Pathway. Molecules. 2015, 20, 5714-5728.

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20. Ardail, D.; Privat, J.; Egret-Charlier, M.; Levrat, C.; Lerme, F.; Louisot, P.

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cells. Pestic. Biochem. Physiol. 2015, 119, 81-89.

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Apoptosis. Structure of Cytochrome c-cardiolipin complex. Biochemistry

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(Moscow). 2013, 78, 1086-1097.

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23. Qi, H.; Fu, H.; Dong, X.; Feng, D.; Li, N.; Wen, C.; Nakamura, Y.; Zhu, B.

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Apoptosis induction is involved in UVA-induced autolysis in sea cucumber

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Stichopus japonicus. J. Photochem. Photobiol., B. 2016, 158, 130-135 .

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24. Orrenius, S.; Zhivotovsky, B. Cardiolipin oxidation sets cytochrome c free. Nat.Chem. Biol. 2005, 1, 188-189. 25. Shi, Y. A structural view of mitochondria-mediated apoptosis. Nat. Struct. Biol. 2001, 8, 394-401.

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26. Zhang, H.; Huang, Q.; Ke, N.; Matsuyama, S.; Hammock, B.; Godzik, A.; Reed,

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conservation of cell death mechanisms. J. Biol. Chem. 2000, 274, 27303-27306.

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27. Scorrano, L.; Korsmeyer, S.J. Mechanisms of cytochrome c release by

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proapoptotic BCL-2 family members. Biochem. Biophys. Res. Commun. 2003,

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Cho, C.K.; Kang, S.; Bae, S.; Lee, Y.S.; Chung, H.Y.; Lee, S.J. Phytosphingosine

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29. Rajah, T.; Chow, S.C. The inhibition of human T cell proliferation by the caspase

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Pharmacol. 2014. 278. 100–106.

423 424 425

Figure 1. Cytochrome C immunostaining of sea cucumber at the epidermis edge of

426

body wall tissues before (a) and after UV exposure for half hour and held 20 C°

427

for 0 hour (b), 1 hour (c), 2 hour (d), 4 hour (e), 6 hour (f) with magnification of

428

200X.

429

Figure 2. Cytochrome C immunostaining of sea cucumber before (a) and after UV

430

exposure for half hour (b-f) at the inner of body wall tissues with magnification

431

of 400X. Cytochrome C immunostaining of sea cucumber body wall tissue at 0

432

hour (b), 1 hour (c), 2 hour (d), 4 hour (e), 6 hour (f) after UV exposure for half

433

hour.

434

Figure 3. Depolarization of mitochondrial transmembrane potential in coelomic fluid

435

cells. Sea cucumbers exposed to UV light for half hour and then left in dark at

436

20 °C for 0 to 6 hours. The coelomic fluid cells were isolated and stained with

437

JC-1, followed by analysis with a flow cytometer. The ratio of red to green

438

fluorescence intensity was presented as means ± SD values of three experiments

439

in triplicate (A). Values in the same dose of different groups with different letters

20


Page 20 of 43

Page 21 of 43


440

(a and b) are significantly different at p < 0.01; Intracellular JC-1 accumulation

441

was observed by fluorescent microscopy (10×) (B).

442

Figure 4. ROS production in the coelomic fluid cells of the sea cucumbers after UV

443

exposure. Sea cucumbers exposed to UV for half hour and then left in dark at

444

20 °C for 0 to 6 hours. The coelomic fluid cells were subsequently extracted and

445

stained by DCFH-DA. (A) Laser confocal microscopy image of coelomic fluid

446

cells stained with ROS detecting probes. (B) Quantification of fluorescence

447

intensity using the Image J software. Values in the same dose of different groups

448

with different letters (a and c) are significantly different at p < 0.01.

449

Figure 5. The sea cucumbers were exposed to UV for half hour and left in room

450

temperature for 0, 1, 2, 4 and 6 hours, then the coelomic fluid cells were isolated

451

immediately for western blotting analysis. (A), The expression of Bax and Bcl-2

452

after UV exposure. COX IV is used as internal control for mitochondrial fraction

453

and β-actin for cytosolic fraction. (B and C), The densitometric analysis of the

454

blots for Bax and Bcl-2 in mitochondria. The quantification result of Bax and

455

Bcl-2 is presented as Bax/Bcl2 ratio (D). Values in the same dose of different

456

groups with different letters (a and b) are significantly different at p < 0.01.

457

Figure 6. Caspase-3 activity changes in sea cucumber intestinal cells (A) and

458

coelomic fluid cells (B) after sea cucumbers exposed to UV radiation.

459

UV+Z-VAD-FMK group sea cucumbers were injected with pan-caspase inhibitor

460

Z-VAD-FMK from cloacal aperture followed by UV exposure as in UV group.

461

Normal group sea cucumbers were placed at same environment for 30 min and 21



462

then left in dark at 20 °C. Results are expressed as n-fold increase in caspase

463

activity compared to the controls. Values in the same dose of different groups

464

with different letters (a to f) are significantly different at p < 0.01.

465


466


467


468


469

Normal group sea cucumbers were placed at same environment for 30 min and

470


471


472


473

Figure 8. The morphological change of sea cucumber melting. UV group sea

474

cucumbers were induced by UV irradiated in 0.056mw/cm2 for 30 min, then left

475

in dark at 20 °C. UV+Z-VAD-FMK group sea cucumbers were injected with

476

pan-caspase inhibitor Z-VAD-FMK from cloacal aperture followed by UV

477

exposure as in UV group. Normal group sea cucumbers were placed at same

478

environment for 30 min and then left in dark at 20 °C.

22


Page 22 of 43

Page 23 of 43


479

Figure 1

480 a

b

c

d

e

f

481 482 Cyto c

483 484 485 486 487

Cyto c

488 489 490 491 492 493 494 495 496 497 498 499 500 501 23



502

Page 24 of 43

Figure 2

503

a

b

c

d

e

f

504 505 506

Cyto c

507 508 509 Cyto c

510 511

24


Page 25 of 43

512


Figure 3

A

B

Fresh

0h

1h

2h

4h

6h

Green

Red

Green

Red

513

25



514

Figure 4 A

B

Fresh

0h

2h

4h

1h

6h

515 516

26


Page 26 of 43

Page 27 of 43


517 518

Figure 5 A

519 520

fresh

0h

1h

2h

4h

6h

521 β-actin 522 523 524 Bax (Cyt.)525 526 Bax (Mit.)527 528 529 530 Bcl-2 (Mit.) 531 532 COX IV533 534 535

B

C

D

536 537 538 539 540

27



541 542

Figure 6

A

543 544

B

545

28


Page 28 of 43

Page 29 of 43


546

Figure 7

A

547 548

B

549

29



550

Page 30 of 43

Figure 8 Fresh

2h

6h

20h

47h

Normal

UV+Z-VAD-FMK

Body wall melting

UV

551

30


Page 31 of 43

552 553


TOC Graphic Inactivation Caspase 9↓

Caspase 3↓

UV

ROS ↑

Pan-caspase Inhibitor

Bcl-2/Bax↓

∆Ψm↓

Mitochondria

Melting Apoptosis UV

Cyto c↑

Activation Caspase 9↑

554 555

31


Caspase 3↑

20 h later


Graphics for Manuscript

1 2 3

Figure 1. Cytochrome C immunostaining of sea cucumber at the epidermis edge of

4

body wall tissues before (a) and after UV exposure for half hour and held 20 C°

5

for 0 hour (b), 1 hour (c), 2 hour (d), 4 hour (e), 6 hour (f) with magnification of

6

200X.

7

Figure 2. Cytochrome C immunostaining of sea cucumber before (a) and after UV

8

exposure for half hour (b-f) at the inner of body wall tissues with magnification

9

of 400X. Cytochrome C immunostaining of sea cucumber body wall tissue at 0

10

hour (b), 1 hour (c), 2 hour (d), 4 hour (e), 6 hour (f) after UV exposure for half

11

hour.

12

Figure 3. Depolarization of mitochondrial transmembrane potential in coelomic fluid

13

cells. Sea cucumbers exposed to UV light for half hour and then left in dark at

14

20 °C for 0 to 6 hours. The coelomic fluid cells were isolated and stained with

15

JC-1, followed by analysis with a flow cytometer. The ratio of red to green

16

fluorescence intensity was presented as means ± SD values of three experiments

17

in triplicate (A). Values in the same dose of different groups with different letters

18

(a and b) are significantly different at p < 0.01; Intracellular JC-1 accumulation

19

was observed by fluorescent microscopy (10×) (B).

20

Figure 4. ROS production in the coelomic fluid cells of the sea cucumbers after UV

21

exposure. Sea cucumbers exposed to UV for half hour and then left in dark at

22

20 °C for 0 to 6 hours. The coelomic fluid cells were subsequently extracted and

23

stained by DCFH-DA. (A) Laser confocal microscopy image of coelomic fluid 1


Page 32 of 43

Page 33 of 43


24

cells stained with ROS detecting probes. (B) Quantification of fluorescence

25

intensity using the Image J software. Values in the same dose of different groups

26

with different letters (a and c) are significantly different at p < 0.01.

27

Figure 5. The sea cucumbers were exposed to UV for half hour and left in room

28

temperature for 0, 1, 2, 4 and 6 hours, then the coelomic fluid cells were isolated

29

immediately for western blotting analysis. (A), The expression of Bax and Bcl-2

30

after UV exposure. COX IV is used as internal control for mitochondrial fraction

31

and β-actin for cytosolic fraction. (B and C), The densitometric analysis of the

32

blots for Bax and Bcl-2 in mitochondria. The quantification result of Bax and

33

Bcl-2 is presented as Bax/Bcl2 ratio (D). Values in the same dose of different

34

groups with different letters (a and b) are significantly different at p < 0.01.

35


36


37


38


39


40


41


42


43


44


45

UV+Z-VAD-FMK group sea cucumbers were injected with pan-caspase inhibitor 2



46


47


48


49


50


51

Figure 8. The morphological change of sea cucumber melting. UV group sea

52

cucumbers were induced by UV irradiated in 0.056mw/cm2 for 30 min, then left

53

in dark at 20 °C. UV+Z-VAD-FMK group sea cucumbers were injected with

54

pan-caspase inhibitor Z-VAD-FMK from cloacal aperture followed by UV

55

exposure as in UV group. Normal group sea cucumbers were placed at same

56

environment for 30 min and then left in dark at 20 °C.

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 3


Page 34 of 43

Page 35 of 43

79


Figure 1

80

a

b

c

d

e

f

81 82 Cyto c

83 84 85 86 87

Cyto c

88 89 90 91 92 93 94 95 96 97 98 99 100 101 4



102

Page 36 of 43

Figure 2

103

a

b

c

d

e

f

104 105 106

Cyto c

107 108 109 Cyto c

110 111

5


Page 37 of 43

112


Figure 3

A

B

Fresh

0h

1h

2h

4h

6h

Green

Red

Green

Red

113

6



114

Figure 4 A

B

Fresh

0h

2h

4h

1h

6h

115 116

7


Page 38 of 43

Page 39 of 43

117 118


Figure 5 A

119 120

fresh

0h

1h

2h

4h

6h

121 β-actin 122 123 124 Bax (Cyt.)125 126 Bax (Mit.)127 128 129 130 Bcl-2 (Mit.) 131 132 COX IV133 134 135

B

C

D

136 137 138 139 140

8



141 142

Figure 6

A

143 144

B

145

9


Page 40 of 43

Page 41 of 43

146


Figure 7

A

147 148

B

149

10



150

Page 42 of 43

Figure 8 Fresh

2h

6h

20h

47h

Normal

UV+Z-VAD-FMK

Body wall melting

UV

151

11


Page 43 of 43


TOC Graphic

Inactivation Caspase 9↓

Caspase 3↓

UV

ROS ↑

Bcl-2/Bax↓

ΔΨm↓

Pan-caspase Inhibitor Mitochondria

Melting Apoptosis UV

Cyto c↑

Activation Caspase 9↑

12


Caspase 3↑

20 h later

Ultraviolet-Ray-Induced Sea Cucumber (Stichopus japonicus) Melting

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