Defining Cancer Cell Bioenergetic Profiles Using a Dual Organelle

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Defining cancer cell bioenergetic profiles using a dual organelle-oriented chemosensor responsive to pH values and electropotential changes Zhongwei Xue, Hu Zhao, Jian Liu, Jiahuai Han, and Shoufa Han Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01934 • Publication Date (Web): 19 Jun 2017 Downloaded from http://pubs.acs.org on June 20, 2017

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Analytical Chemistry

Defining cancer cell bioenergetic profiles using a dual organelleoriented chemosensor responsive to pH values and electropotential changes

Zhongwei Xue,a Hu Zhao,a Jian Liu, a Jiahuai Han,b and Shoufa Hana,* a

Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key

Laboratory for Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, and Innovation Center for Cell Signaling Network, Xiamen University; bState key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, 361005, China

Corresponding author *To whom correspondence should be addressed. E-mail: [email protected]. Fax: +865922181728. Xiamen University.

ACS Paragon Plus Environment

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Abstract Cell fate is largely shaped by combined activity of different types of organelles, which often feature functionally critical parameters succumb to pathological inducers. We herein report the analysis of cell bioenergetic profiles with a dual organelle-oriented chemosensor (RC-AMI), partitioning in mitochondria to give blue fluorescence and in lysosomes to give red fluorescence. Responsive to lysosomal pH and mitochondrial transmembrane potential (∆Ψm), two parameters crucial to cell bioenergetics, RC-AMI enables dual colored reporting of lysosomal acidity and ∆Ψm, revealing upregulated ∆Ψm and imbalance dramatically shifted favoring ∆Ψm over lysosomal acidity in cancer cells whereas the tendency is reversed in starved cells. Complementing classical homo-organelle specific sensors, this dual organelle-oriented and fluorescently responsive probe offers a new tool to detect imbalance between lysosomal acidity and mitochondrial ∆Ψm, an index critical for cancer bioenergetics.

Keywords:

Cancer bioenergetics, organelle-oriented, fluorescence imaging, lysosomal pH,

mitochondrial electrical potential


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Introduction Mammalian cells harbour distinct kinds of organelles functionally essential for cell performance. Mitochondria are bioenergetic and signaling organelles relying on negative transmembrane electrical potential (∆Ψm) for energy production.1,2 Mitochondria are integral in stress sensing and pertinent to myriad pathological events including tumorigenesis.3 Attenuated ∆Ψm triggers diverse cellular events ranging from autophagy to cell death.4 Lysosomes are ubiquitous digestive organelles, and lysosomal acidity is critical for cell homeostasis, and immunity, etc.5 Aberrant lysosomal pH has been manifested in multiple pathological events including cancers and lysosomal storage diseases.6 Given the distinct roles of lysosomes and mitochondria in regulating cell metabolism,3,7,8 it is imperative to define off-balance of lysosomal pH and ∆Ψm in stressed cells or cancers. Homo-organelle homing dyes are widely used for bioimaging.9 However acidotropic lysosomal sensors and ∆Ψm-tropic mitochondrial sensors quickly dissipate upon loss of ∆Ψm or lysosomal acidity, and thus are often incapable of assessing abnormal organelles. Apart from organelle parameters, organelle biogenesis, dynamically regulated and functionally significant,10 is difficult to probe with conventional organelle-specific dyes. Lysosomes and mitochondria are highly dynamic and critical for cell bioenergetics. Imbalance between lysosomal pH and ∆Ψm in pathological cells is challenging to explore with conventional organelle sensors. We herein report the profiling of intracellular lysosomal acidity over ∆Ψm with RC-AMI which simultaneously partitions in lysosomes and mitochondria, driven by lysosomal acidity and ∆Ψm (Scheme 1). RC-AMI consists of an “always-on” blue emissive coumarin fluorophore, an acidity-activatable rhodamine-lactam profluorophore, and a ∆Ψm-responsive quaternary ammonium ion (AMI) linker. Exhibiting red cscence in acidic lysosomes and blue fluorescence


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in alkaline mitochondria, RC-AMI effectively differentiates the levels of lysosomal acidity and ∆Ψm in cancer cells and stressed cells, and also the imbalance between these two organelle parameters critical for cell bioenergetic status. Results and discussion Mitochondria and lysosomes are critical regulators of cell bioenergetics. We recently reported the detection of mitochondrial depolarization with triphenylphosphonium (TPP)conjugated diad of rhodamine-lactam/coumarin (RC-TPP), which selectively accumulates in mitochondria, and relocates into lysosomes upon loss of ∆Ψm.11 Cationic TPP moiety efficiently ferries various cargoes into mitochondria.12-14 We envisioned that integration of a lysosomal sensor with a ∆Ψm responsive entity of attenuated affinity relative to TPP might afford a dual organelle-partitioned sensor capable of simultaneously reporting ∆Ψm and lysosomal pH. Hence RC-AMI with a cationic quaternary ammonium linker was designed for analysing lysosomal acidity and ∆Ψm in cells. pH dependent fluorescence of RC-AMI Rhodamine-lactams, a group of nonfluorescent rhodamine derivatives featuring intramolecular lactam, are poised to proton-triggered isomerization to give fluorescent rhodamine species and have been employed for lysosomal imaging.15-19 As such, RC-AMI was designed to encompass a coumarin (CM) fluorophore, ∆Ψm-responsive ammonium linker, and rhodamine-X-lactam (ROX) which is highly sensitive to lysosomal acidity.20 pH titration shows that RC-AMI exhibits bright rhodamine fluorescence in acidic media, which intensifies as pH decreases (pH 6.5-4.5). The “switch-on” rhodamine fluorescence is consistent with proton mediated fluorogenic opening of the intramolecular lactam (Figure 2A). As expected, RC-AMI


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displays negligible rhodamine fluorescence in alkaline media (pH 7.2-8.5), and “always-on” coumarin fluorescence which decreases in acidic pH (Figure 1B-C). The pH dependent blue to red fluorescence patterns of RC-AMI match pH windows of mitochondria (∼pH 8.0) and lysosomes (pH 6.0-4.5) (Figure 1D), suggesting its applicability to optically differentiate lysosomes and mitochondria. Dual organelle-oriented partition and fluorescence of RC-AMI in live cells To ascertain its subcellular distribution, RC-AMI was pulsed with HeLa cells expressing Green Fluorescent Protein tagged Tom20 (Tom20-GFP), which is an constituent protein of mitochondria used for mitochondria staging. Confocal microscopic analysis reveals abundant coumarin fluorescence colocalized with GFP in Tom20-GFP+ cells (Figure 2A), showing that RC-AMI effectively accumulates in mitochondria and gives blue fluorescence. In parallel, punctate rhodamine fluorescence was observed to eclipse GFP signals in HeLa cells expressing Lamp2-GFP (GFP-tagged lysosome associated membrane protein 2) (Figure 2B), proving accumulation and rhodamine fluorescence activation of RC-AMI in lysosomes. We next probed the structural factor conferring simultaneous accumulation of RC-AMI in mitochondria and lysosomes. HeLa cells cultivated with varied doses of RC-AMI display relatively constant coumarin to rhodamine (CM/ROX) fluorescence ratios in the range of 1.5-2.0 (Figure 3). In line with reported studies,11 RC-TPP selectively accumulates in mitochondria with high CM/ROX fluorescence ratios around 25, whereas rhodamine-coumarin (RC), lacking AMI moiety, is exclusively located in lysosomes (Figure 3C, Supporting information, Figure S1-S). These findings validate the use of AMI as a ∆Ψm-responsive entity with attenuated affinity as


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compared to TPP, and feasibility of AMI-integrated lysosomal sensors for simultaneous partition in mitochondria and lysosomes. Lysosomal acidity and ∆Ψm mediated intra-organelle partition and dual colored fluorescence of RC-AMI We proceeded to ascertain cellular factors mediating organelle accumulation of RC-AMI. HeLa cells were first treated with RC-AMI in the presence of Bafilomycin A1 (BFA) or nigericin. BFA selectively inhibits vacuolar ATPase-H1 pump and neutralizes lysosomes.21 Nigericin is a H+/K+ ionophore which homogenizes pH gradients inside cells while increasing mitochondrial ∆Ψm.22,23. Consistently, no rhodamine signals could be identified in BFA- or nigericin-treated cells owing to loss of lysosomal acidity. Meanwhile, nigericin-treated cells display markedly enhanced coumarin fluorescence relative to control cells (Figure 4). These results prove lysosomal acidity dependent activation of rhodamine fluorescence of RC-AMI. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) effectively suppress ∆Ψm.24,25 HeLa cells treated with CCCP display attenuated coumarin fluorescence and decreased CM/ROX signals (Figure 4), showing ∆Ψm mediated mitochondrial accumulation of RC-AMI. Taken together, these cell imaging studies show that RC-AMI undergoes ∆Ψm and acidity mediated partition in mitochondria and lysosomes. Autophagy is a catabolic mechanism involving lysosomal degradation of cellular components in response to cell stress such as starvation.26 To evaluate organelle responses in starvation, HeLa cells were cultivated in Hanks’ balanced salt solution (HBSS) free of amino acids to starve cells. HBSS-treated cells display dramatically enhanced rhodamine fluorescence decreased CM/ROX fluorescence ratio (Figure 4, Supporting information, Figure S3), indicative of upregulated


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lysosomal acidity over ∆Ψm under catabolic settings (Figure 4), which is consistent with nutrient deprivation induced lysosomal acidification.26 The discern of altered fluorescence intensity and ratios of CM/ROX in cells proves the usefulness of RC-AMI to monitor changes and imbalance of organelle parameters in response to exogenous stimuli. Assessing bioenergetic profiles of cancer cells Abnormal metabolism is a hallmark of cancers.27-29 Particularly, mitochondrial metabolism is necessary for tumorigenesis and cancer cell proliferation.30,31 To probe cell type specific bioenergetic profiles, RC-AMI was pulsed with Chinese Hamster Ovary cells (CHO), mouse fibroblast cells (L929), mouse embryonic fibroblast cells (3T3), human breast adenocarcinoma cells (MCF-7), human fibrosarcoma cells (HT-1080), and human cervical HeLa cells. Abundant coumarin fluorescence outside lysosomes was observed in cancer cell lines of HeLa, MCF-7, and HT-1080 cells over normal cell lines of CHO, 3T3, and L929 cells (Figure 5A), revealing high levels of ∆Ψm in these examined cancer cells. The weak ∆Ψm in L929, 3T3 and CHO cells were also confirmed by cell staining with rhodamine 123, a commercial ∆Ψm-responsive dye widely used for mitochondria imaging (Figure S3, Supporting information). In addition, HT1080 cells display high lysosomal acidity relative to HeLa and MCF-7 cells, as evidenced by the intense rhodamine signals inside cells (Figure 5A), indicating cell line specific lysosomal acidification or upregulated biogenesis. Quantitative flow cytometry analysis reveals dramatically elevated CM/ROX fluorescence ratios in cancerous cell populations over normal cell lines (Figure 5B, Supporting information, Figure S4), proving the utility of RC-AMI to detect imbalance between ∆Ψm and lysosomal acidity in a broad spectrum of cell lines. Complementing mono-organelle specific optical systems,32-37 RC-AMI offers a new tool to simultaneously monitor the status of lysosomes and mitochondria, which are critical regulators of cell bioenergetics.


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Conclusions Defining organelle alterations in diseased cells is of use to decipher the roles of organelles in pathology. Cancers are hallmarked by reprogrammed metabolism, of which lysosomes and mitochondria are key regulators of cancer bioenergetics.27-29 Responsive to lysosomal acidity and ∆Ψm, RC-AMI simultaneously partitions in lysosomes and mitochondria. Being blue emissive in mitochondria and red emissive in lysosomes, RC-AMI enables sensitive dual colored tracking of alteration of lysosomal acidity and ∆Ψm in stressed cells and cancer cells. Off-balance of lysosomal pH over ∆Ψm in cells could be monitored by fluorescence ratios of coumarin to rhodamine, providing a straightforward approach to assess bioenergetic profiles of diverse cells. Complementing existing approaches to detect metabolites and metabolic activities which have advanced our understanding of cancers,38 RC-AMI represents a new perspective from which to deconvolute metabolic roles of lysosomes and mitochondria in cancers, and potentially to image organelle responses to therapeutics against cancer metabolism. Experimental procedure Materials and Method: Bafilomycin A1 (BFA) was purchased from Selleck. Rapamycin, carbonyl cyanide m-chlorophenylhydrazone (CCCP) was obtained from Sigma. CellTiter 96® AQueous One Solution Cell Proliferation Assay reagent was obtained from Promega RC-TPP and RC were synthesized according to published procedures.18 All other chemicals were obtained from Alfa-Aesar unless otherwise specified. Cell culturing: HeLa, L929, CHO, HT-1080, 3T3 and MCF-7 cells were obtained from American Type Culture Collection (ATCC). Transient transfection of HeLa cells was performed using calcium phosphate method. Lentiviral infection was used for stable expression.


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Recombinant lentiviruses were packaged in 293T cells in the presence of helper plasmids (pMDLg, pRSV-REWV and pVSV-G) using a calcium phosphate precipitation method. The transfected cells were cultured for 48 h and the viruses were then collected for infection. All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU penicillin, and 100 mg/ml streptomycin at 37 °C in a humidified incubator containing 5% CO2. Gene expression: full-length cDNA of Lamp2 were cloned into BamHI and XhoI sites of the lentiviral vector pBOB-GFP using the Exo III-assisted ligase-free cloning method. All plasmids were verified by DNA sequencing. The details of the sequences are available upon request. For lentivirus production, HEK293T cells were transfected by the calcium phosphate precipitation method. The virus-containing medium was harvested 36-48 h later and was added to HeLa cells. Fluorescence spectra and confocal fluorescence imaging: The fluorescence spectra of RCAMI were performed on SpectraMax M5. Confocal fluorescence microscopic imaging was performed on Zeiss LSM 780 using the following filters: λex = 405 nm and λem = 410-490 nm for coumarin, λex = 488 nm and λem = 490-553 nm for GFP, λex = 565 nm and λem = 593-735 nm for rhodamine. The fluorescence of rhodamine and coumarin in cells was respectively shown in red and blue in the figures while fluorescence of GFP in cells was shown in green. Images of the fluorescence RC-AMI and that of GFP in cells were merged using Photoshop CS6. Quantitative imaging analysis was carried out on unprocessed images using ImageJ software. Graph was generated by GraphPad Prism5 and origin 8.0 software. Synthesis of RC-AMI (Scheme S1, Supporting Inofrmation): To a flask containing RC (0.36 g, 0.44 mmol), K2CO3 (0.36 g) and dichloromethane (10 ml) was added iodomethane (1.2g, 8.5


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mmol). The solution was stirred at rt fro 3 h and then concentrated to remove the solvent and excess iodomethane. The residue was purified by silica gel column chromatography using DCM/ MeOH /TEA (20:1:1, v/v/v) as the eluent to afford RC-AMI (0.30 g, 0.3 mmol) in 68 % yield. 1

H-NMR (500 MHz, CD3OD), δ:8.58 (1 H, s), 7.87 (1 H, d, J 7.0), 7.62 – 7.51 (3 H, m), 7.09 (1

H, d, J 7.4), 6.83 (1 H, dd, J 9.1, 2.3), 6.54 (1 H, d, J 2.1), 5.95 (2 H, s), 3.56 (8 H, dq, J 21.0, 7.1), 3.39 (2 H, t, J 7.0), 3.14 (4 H, t, J 5.5), 3.12 – 3.01 (12 H, m), 2.94 – 2.81 (4 H, m), 2.56 – 2.47 (2 H, m), 2.47 – 2.38 (2 H, m), 2.04 – 1.95 (4 H, m), 1.87 – 1.79 (4 H, m), 1.24 (6 H, t, J 7.1);

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C-NMR (126 MHz, CD3OD):169.75, 165.75, 163.78, 159.33, 154.86, 154.47, 149.89,

149.60, 145.51, 134.62, 132.80, 131.69, 130.84, 129.81, 125.41, 125.30, 123.77, 119.47, 111.86, 109.38, 109.11, 105.56, 97.29, 67.86, 63.68, 62.06, 51.87, 50.85, 50.39, 46.05, 34.74, 34.25, 28.25, 23.02, 22.46, 22.33, 12.73. HRMS (C52H59N6O5+): calculated (M+): 847.4541, found: 847.4569. pH titration of RC-AMI: Aliquots of the stock solution of RC-AMI in DMSO was added to Tris-HCl buffer containing 40% MeOH of various pH values (4.03, 4.19, 4.41, 4.51, 4.62, 4.71, 4.81, 4.89, 4.99, 5.10, 5.20, 5.29, 5.39, 5.49, 5.61, 5.70, 5.79, 5.90, 5.98, 6.10, 6.2, 6.32, 6.41, 6.52, 6.99, 7.51, 7.98, 8.50) to a final concentration of 10 µM. The fluorescence emission spectra were recorded as a function of pH using λex = 430 nm for coumarin and λex = 585 nm for ROX. Subcellular distribution of RC-AMI in living cells. Lam2-GPF+ HeLa cells and Tom20GPF+ HeLa cells were respectively seeded on 35 mm glass-bottom dishes (NEST) and incubated in DMEM for 24 h, followed by addition of RC-AMI (3 µM) for 30 min. The cells were rinsed with fresh DMEM for 3 times and then analyzed with a confocal fluorescence microscope.


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∆Ψm and lysosomal acidity dependent organelle partition of RC-AMI. HeLa cells prestained with RC-AMI (3 µM) were incubated for 6 h in HBSS or DMEM spiked with BFA (100 nM), CCCP (20 µM), nigericin (2 µM), or no addition. The cells were rinsed with fresh DMEM for 3 times and then analyzed with a confocal fluorescence microscope and flow cytometry. Effects of sensor concentrations on organelle partition. HeLa cells were respectively incubated for 30 min in DMEM spiked with RC-AMI (1, 3, 6 µM), RC-TPP (1, 3, 6 µM), or RC (1, 3, 6 µM). The cells were then respectively rinsed with fresh DMEM for 3 times and then analyzed by confocal fluorescence microscopy and flow cytometry. For flow cytometry analysis, the fluorescence emission intensity of coumarin was recorded by FL1 filter (430-470 nm) using excitation wavelength of 405 nm while that of ROX was recorded by FL2 filter (590-630 nm) using excitation wavelength of 561 nm. 10000 cells were were gated under identical conditions, analyzed and the data were processed by GraphPad Prism5. Analysis of ∆Ψm and lysosomal acidity in diferent cell lines with RC-AMI: HeLa, L929, CHO, 3T3, HT-1080 and MCF-7 cells were respectively incubated for 30 min in DMEM containing RC-AMI (3 µM) or rhodamine 123 (3 µM). The cells were further incubated in fresh DMEM for 10 min and then analyzed by confocal fluorescence microscopy and flow cytometry. Cytotoxicity of RC-AMI: The cytotoxicity of RC-AMI was evaluated on HeLa cells. The cells were cultured with DMEM medium containing RC-AMI (0, 1, 2.5, 5, 10 µM) for for various amount of time (6-48 h) at 37 °C with 5% CO2. Cell number and cell viability were determined using CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS assay) .


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Acknowledgements This work was supported by grants from, NSF China (21572189, 21602185, 21272196), 973 program 2013CB933901, the Fundamental Research Funds for the Central Universities (20720160052, 20720150047); Natural Science Foundation of Fujian Province of China (2016J05047), Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT 13036) and Foundation for Innovative Research Groups of the National Natural Science Foundation of China (Grant No. 21521004). Dr. J. Han was supported by grants from NSF China (91429301, 31420103910, 31330047, 31221065), the National Scientific and Technological Major Project (2013ZX10002-002), the Hi-Tech Research and Development Program of China (863program; 2012AA02A201).

Supporting Information Supporting information on cellular biodistribution of RC and RC-TPP; flow cytometry analysis of cells stained with RC-AMI, and cytotoxicity of RC-AMI.


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FIGURES

"Always-on" blue fluorescence

Rhodamine-lactam

A

N

Acidity responsive; "turn-on" fluorescence; lysosome targeting

O

N O

O

N

H N

N N

O

Coumarin

O

AMI Potential responsive linker

B H+ N

O

H+

N

-OH

H+

N

O

N

O

O N H

N

N

NH O

O

N

-OH

N

H N

O

O

N

O

O

Mitochondrion

Lysosome

Scheme 1. Profiling lysosomal acidity and mitochondrial ∆Ψm by dual organelle-partitioned RC-AMI. (A) Chemical structure of RC-AMI, which contains a coumarin fluorophore and acidresponsive rhodamine-lactam bridged via a cationic quaternary ammonium linker. (B) RC-AMI undergoes acidity mediated accumulation and fluorescence activation in lysosomes, and ∆Ψm mediated accumulation in alkaline mitochondria to give blue fluorescence.


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A N

O

N

N N

N

H N

O

Lactam form Blue fluorescence

O

O

N

Protonation O

Neutralization

O

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N O

O N H

N

NH O

O

N

Amide form Intense red fluorescence

Figure 1. pH dependent dual colored fluorescence of RC-AMI. A) Proton mediated fluorogenic isomerization of RC-AMI gives highly fluorescent rhodamine species. B) Coumarin fluorescence emission of RC-AMI (20 µM) at pH 4.0-8.5 (λex = 430 nm). C) Rhodamine fluorescence emission of RC-AMI (10 µM) (λex = 585 nm). D) pH titration curves of RC-AMI (20 µM); fluorescence emission of rhodamine (λem = 605 nm) and coumarin (λem = 475 nm) were plotted over buffer pH; E) pH dependent fluorescence ratios of rhodamine (I605 nm) to coumarin (I475 nm).


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Figure 2. Partition and fluorescence of RC-AMI in mitochondria and lysosomes. Tom20-GFP+ (A) or Lamp2-GFP+ HeLa cells (B) stained with RC-AMI (3 µM) were visualized by confocal fluorescence microscopy. Colocalization of coumarin with green fluorescence is shown in cyan. Plots of fluorescence of coumarin, rhodamine, and GFP were measured along the lines shown in zoomed images, which reveal coumarin colocalized with Tom20-GFP in mitochondria and rhodamine colocalzied with Lamp2-GFP in lysosomes. Bar, 10 µm.


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A

Coumarin

Rhodamine

Bright view

Merge

1 µM

3 µM

6 µM

B 100

% of Max

% of Max

100 80 60 40

0

80 60 40 20

20

0

101 102 103 104 105

Rhodamine fluorescence 33

RC-AMI

22 11 00

11 33 6 6 Concentration C o n c e n t r a t i o n(µ ( µM) M)

Fluorescence ratio R a t io ( C M / R O X ) (CM/ROX)

C

101 102 103 104 105

CoumarinRfluorescence C -T P P

R C -A M I

Fluorescence ratio R a t io ( C M / R O X ) (CM/ROX)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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40 40

RC-TPP

30 30 20 20 10 10 00

11 33 66 Concentration (µM)

C o n c e n t r a t io n ( µ M )

Figure 3. AMI mediated partition of RC-AMI in mitochondria and lysosomes. HeLa cells cultivated with RC-AMI (1-6 µM) were analysed by confocal fluorescence microscopy (A) or flow cytometry (B). CM/ROX fluorescence ratios in cells stained with RC-AMI or RC-TPP (control) were quantitated by flow cytometry and plotted as a function of probe concentrations (0, 1, 3, 6 µM) indicated by corresponding colored curves in the insert. (C). Error bars represent standard deviation of 10000 cells.


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Figure 4. Alteration of lysosomal acidity and ∆Ψm detected by RC-AMI. (A) HeLa cells were stained with RC-AMI in glucose-containing HBSS free of amino acids, or in DMEM supplemented with BFA, nigericin, CCCP, or no addition before confocal microscopic analysis. Bar, 10 µm. (B) CM/ROX fluorescence ratios in HeLa cells in response to indicated stimuli were determined by flow cytometry. Error bars represent standard deviation of 10000 cells.


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Figure 5. Imbalance of ∆Ψm over lysosomal acidity in cancer cells determined with RC-AMI. (A) Confocal fluorescence microscopic images of different cell lines stained with RC-AMI. Bar, 10 µm. (B) Intracellular CM/ROX fluorescence ratios were determined by flow cytometry and plotted over cell lines. Error bars represent standard deviation of 10000 cells.


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Defining Cancer Cell Bioenergetic Profiles Using a Dual Organelle

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