Secreted Frizzled-Related Protein 4
2010; Elsevier BV; Volume: 176; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2010.090465
ISSN1525-2191
AutoresAjit Muley, Syamantak Majumder, Gopi K. Kolluru, Steve Parkinson, Helena M. Viola, Livia C. Hool, Frank Arfuso, Ruth Ganß, Arun Dharmarajan, Suvro Chatterjee,
Tópico(s)Kruppel-like factors research
ResumoWnt signaling is involved in developmental processes, cell proliferation, and cell migration. Secreted frizzled-related protein 4 (sFRP4) has been demonstrated to be a Wnt antagonist; however, its effects on endothelial cell migration and angiogenesis have not yet been reported. Using various in vitro assays, we show that sFRP4 inhibits endothelial cell migration and the development of sprouts and pseudopodia as well as disrupts the stability of endothelial rings in addition to inhibiting proliferation. sFRP4 interfered with endothelial cell functions by antagonizing the canonical Wnt/β-catenin signaling pathway and the Wnt/planar cell polarity pathway. Furthermore, sFRP4 blocked the effect of vascular endothelial growth factor on endothelial cells. sFRP4 also selectively induced apoptotic events in endothelial cells by increasing cellular levels of reactive oxygen species. In vivo assays demonstrated a reduction in vascularity after sFRP4 treatment. Most importantly, sFRP4 restricted tumor growth in mice by interfering with endothelial cell function. The data demonstrate sFRP4 to be a potent angiogenesis inhibitor that warrants further investigation as a therapeutic agent in the control of angiogenesis-associated pathology. Wnt signaling is involved in developmental processes, cell proliferation, and cell migration. Secreted frizzled-related protein 4 (sFRP4) has been demonstrated to be a Wnt antagonist; however, its effects on endothelial cell migration and angiogenesis have not yet been reported. Using various in vitro assays, we show that sFRP4 inhibits endothelial cell migration and the development of sprouts and pseudopodia as well as disrupts the stability of endothelial rings in addition to inhibiting proliferation. sFRP4 interfered with endothelial cell functions by antagonizing the canonical Wnt/β-catenin signaling pathway and the Wnt/planar cell polarity pathway. Furthermore, sFRP4 blocked the effect of vascular endothelial growth factor on endothelial cells. sFRP4 also selectively induced apoptotic events in endothelial cells by increasing cellular levels of reactive oxygen species. In vivo assays demonstrated a reduction in vascularity after sFRP4 treatment. Most importantly, sFRP4 restricted tumor growth in mice by interfering with endothelial cell function. The data demonstrate sFRP4 to be a potent angiogenesis inhibitor that warrants further investigation as a therapeutic agent in the control of angiogenesis-associated pathology. 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73: 213-223Crossref PubMed Scopus (154) Google Scholar Secreted frizzled-related protein 4 (sFRP4) is a member of the secreted frizzled-related protein family of Wnt inhibitors that bind directly to Wnt and antagonize both canonical and noncanonical Wnt pathways.12Kawano Y Kypta R Secreted antagonists of the Wnt signalling pathway.J Cell Sci. 2003; 116: 2627-2634Crossref PubMed Scopus (1282) Google Scholar We have previously shown antiproliferative and proapoptotic roles for sFRP4 during normal homeostasis in tissues such as ovary, corpus luteum, placenta, and mammary gland13White L Suganthini G Friis R Dharmarajan A Charles A Expression of secreted frizzled-related protein 4 in the primate placenta.Reprod Biomed Online. 2009; 18: 104-110Abstract Full Text PDF PubMed Scopus (8) Google Scholar, 14Constantinou T Baumann F Lacher MD Saurer S Friis R Dharmarajan A SFRP-4 abrogates Wnt-3a-induced beta-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of in vitro mammary differentiation.J Mol Signal. 2008; 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The exact mechanisms by which Wnt affects angiogenesi remains poorly understood; however, Wnt signaling and the requirements of the canonical Wnt pathway appear to be essential in endothelial cell (EC) commitment developing from embryonic stem cells.19Wang H Gilner JB Bautch VL Wang DZ Wainwright BJ Kirby SL Patterson C Wnt2 coordinates the commitment of mesoderm to hematopoietic, endothelial, and cardiac lineages in embryoid bodies.J Biol Chem. 2007; 282: 782-791Crossref PubMed Scopus (61) Google Scholar Furthermore, coexpression of Wnt proteins and Wnt pathway inhibitors by endothelial cells is implicated in the regulation of angiogenesis.20Dufourcq P Couffinhal T Ezan J Barandon L Moreau C Daret D Duplaa C FrzA, a secreted frizzled related protein, induced angiogenic response.Circulation. 2002; 106: 3097-3103Crossref PubMed Scopus (90) Google Scholar, 21Goodwin AM Sullivan KM D'Amore PA Cultured endothelial cells display endogenous activation of the canonical Wnt signaling pathway and express multiple ligands, receptors, and secreted modulators of Wnt signaling.Dev Dyn. 2006; 235: 3110-3120Crossref PubMed Scopus (88) Google Scholar To date there are no published data demonstrating the involvement of sFRP4 in angiogenesis. We report our investigations on the role of sFRP4 on EC physiology using a variety of in vitro assays and its effect on physiological and tumor-associated angiogenesis using in vivo models. ECV-304 cells were a donation from V. Shah, GI Research Unit, Mayo Clinic, Rochester, MN; Li EA.hy926 cells were donated by C. Edgel, Tissue Culture Facility, UNC Lineburger Comprehensive Cancer Center, University of North Carolina, Chapel Hill; porcine aortic endothelial cells (PAECs) were donated by R. Rieben, University of Bern, Switzerland. Human umbilical vein endothelial cells (HUVECs) were a donation from Dr Chooi-May Lai, Lions Eye Institute, The University of Western Australia. All experiments were performed using EA.hy926 cells, but crucial experiments were repeated in HUVECs and representative experiments are shown whenever possible. However, results were comparable with all cell lines used. Dulbecco's modified Eagle's medium (DMEM) was purchased from Hi-Media, Mumbai, India. Fetal bovine serum was supplied by Invitrogen Life technologies. Anti– β-catenin antibodies were purchased from Millipore. Matrigel was obtained from BD Biosciences, San Jose, CA. Polyether-polyurethane foam sponge was obtained from Amersham Biosciences (SF) Corp., Piscataway, NJ. All other chemicals were of reagent grade and were obtained commercially. For cell culture, cells were maintained in DMEM supplemented with 10% FBS (v/v), 1% penicillin (w/v) and streptomycin (w/v) at 37°C/5% CO2. sFRP4 was supplied by Upstate (Lake Placid, NY). Experiments using Wistar rats were conducted with the approval of the Animal Ethics Committee of Anna University, Chennai, India. Homozygous female BALB/c nu/nu athymic mice experiments were conducted with approval from The University of Western Australia Animal Ethics Committee. Primary PAECs and 2 immortalized cell lines (ECV-304 and EA.hy926) were cultured in 24-well plates coated in collagen type 2 at 2 × 105 cells per well to produce confluent monolayers. The monolayers were wounded in a line using a standard 100-μl pipette tip and washed with PBS (pH 7.4) to remove cell debris before incubation with different concentrations of sFRP4 (125 and 250 pg/ml). The area of the cell-free wound at selected time points (0 and 8 hours) was recorded using a Nikon digital camera and analyzed using Image J image analysis software (Release α 4.0 3.2). The wound healing effect was calculated as the percentage of remaining cell-free area (at 8 hours) compared with the initial wound area. The reversibility of the sFRP4-mediated effect was studied by washing the cells with PBS 24 hours post treatment. Migration in endothelial cells was examined using a Boyden chamber migration assay. Collagen-treated polycarbonate membranes (pore size 8 μm) were used for the assay. HUVECs were pre-treated with different concentrations of sFRP4 (0 and 125 pg/ml) for 2 hours and trypsinized; 1 × 105 cells were loaded in the upper chamber of the Boyden apparatus with or without sFRP4. After 4 hours of incubation at 37°C and 5%CO2, the nonmigrated cells of the upper chamber were removed using a cotton swab. The migrated cells on the underside were fixed with 4% paraformaldehyde in PBS (pH 7.4) and stained with propidium iodide (1 μg/ml). The migrated cells were counted at ×20 magnification using a fluorescent microscope (Olympus IX71). HUVECs were seeded on collagen-coated 12-well plates (1 × 105 cells per well). After 4 hours of incubation at 37°C/5%CO2, the media was changed to medium supplemented with sFRP4 (0 or 125 pg/ml). The cells were incubated with sFRP4 overnight (12 hours) at 37°C/5%CO2. The endothelial cells formed ring-like structures on overnight incubation. The number of rings formed was counted at ×20 magnification using a brightfield phase contrast microscope. HUVECs were seeded as above (1 × 105 cell per well), but on coverslips coated with collagen type 2 in 12-well plates and incubated for 12 hours at 37°C/5%CO2. The endothelial cells formed ring-like structures on overnight incubation. Each coverslip was mounted on a customized live cell chamber and placed on the microscope stage. Single ring structures were identified and the microscope stage was fixed in place, after which the cells were monitored for up to 15 minutes with or without sFRP4 treatment (125 pg/ml). Images were taken at various time points. EA.hy926 cells (which express the vascular endothelial growth factor [VEGF] receptor) were grown to confluence as per the wound scratch protocol. After wounding, cells were incubated with either VEGF (20 nmol/L), Avastin (125 pg/ml), sFRP4 (125 pg/ml), a combination of VEGF and Avastin, or VEGF and sFRP4 for 12 hours. The amount of wound healing was recorded as previously described. HUVECs were grown to confluence as per wound scratch assay. After wounding, they were washed with PBS (pH 7.4) to remove cell debris before incubation using the GSK-3β inhibitor, LiCl (500 μmol/L; Sigma, St Louis, MO), alone or in combination with sFRP4 (125 pg/ml) for 4 hours. The amount of cell migration was recorded as previously described. The proliferation assay was performed as previously reported.22de Jesus Perez VA Alastalo TP Wu JC Axelrod JD Cooke JP Amieva M Rabinovitch M Bone morphogenetic protein 2 induces pulmonary angiogenesis via Wnt-beta-catenin and Wnt-RhoA-Rac1 pathways.J Cell Biol. 2009; 184: 83-99Crossref PubMed Scopus (143) Google Scholar In summary, HUVECs were seeded at a density of 3000 cells per well in 96 wells and incubated overnight at 37°C/5% CO2, and the following day the cells were treated with sFRP4 (125 pg/ml) in the presence and absence of LiCl (500 μmol/L). The cells were further incubated for 48 hours at 37°C/5%CO2. After incubation the cells were trypsinized and counted in a hemocytometer (Nebauer Improved, Crown Scientific). Calcium levels were determined as previously reported.23Viola HM Arthur PG Hool LC Transient exposure to hydrogen peroxide causes an increase in mitochondria-derived superoxide as a result of sustained alteration in L-type Ca2+ channel function in the absence of apoptosis in ventricular myocytes.Circ Res. 2007; 100: 1036-1044Crossref PubMed Scopus (107) Google Scholar Briefly, intracellular calcium was monitored in HUVECs using the fluorescent indicator Fura-2 acetoxymethyl ester (Fura-2, 1 μmol/L, Molecular Probes). Fluorescence at 340/380 nm excitation and 510 nm emission wavelengths were measured at 1-minute intervals with an exposure of 50 milliseconds on a Hamamatsu Orca ER digital camera attached to an inverted Nikon TE2000-U microscope. Ratiometric 340/380-nm signal of individual HUVECs was quantified using Metamorph 6.3 to measure signal intensity of manually traced cell regions. An equivalent region not containing cells was used for background and was subtracted. Ratiometric 340/380-nm fluorescence was plotted relative to the pretreatment fluorescence and assigned a value of 1.0. Fluorescent ratios recorded for 3 minutes at 37°C just before and the last 3 minutes of a 10-minute exposure to 125 pg/ml sFRP4 were averaged, and increases in fluorescent ratios were reported as a percentage increase from the baseline average. After 10 minutes exposure to 125 pg/ml, a further 125 pg/ml of sFRP4 was added to create a final dose of 250 pg/ml, and the change in fluorescence was monitored as per the 125 pg/ml dose. Whole cell lysates and Western blot analysis were performed as previously described with minor modifications.14Constantinou T Baumann F Lacher MD Saurer S Friis R Dharmarajan A SFRP-4 abrogates Wnt-3a-induced beta-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of in vitro mammary differentiation.J Mol Signal. 2008; 3: 10-23Crossref PubMed Scopus (37) Google Scholar Briefly, HUVECs at 70% confluence were stimulated with LiCl (500 μmol/L; Sigma, St Louis, MO) in DMEM for 30 minutes at 37°C and then sFRP4 (125 pg/ml) or PBS was added and cells incubated for a further 4 hours at 37°C before being harvested for protein. Lysates were prepared by scraping cells in radioimmunoprecipitation assay buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl pH7.5, 1% triton X100, 0.5% sodium deoxycholate, 0.1% SDS and 0.1 mmol/L PMSF) on ice. The protein content of the lysates was normalized after quantification by Bradford protein estimation.24Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (204486) Google Scholar The lysates were boiled in Laemmli buffer for 5 minutes at 90°C before loading onto 10% SDS-PAGE gel. After electrophoresis the proteins were transferred onto a nitrocellulose membrane using wet blotting apparatus (Biorad). The membranes were blocked using 5% nonfat milk in Tris-Buffered Saline Tween-20 (TBST) and probed for β-catenin using anti–β-catenin antibody at 1:1000 dilution in TBST (Cell Signaling) and horse radish peroxidase (HRP)-labeled secondary antibody at a dilution of 1:10,000 in TBST (Pierce). The blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce). β-actin antibody was used at a concentration of 1:5000 (Sigma). The secondary antibody was anti-mouse HRP used at 1:10,000 dilution (Pierce). Isolation of nuclear fragments was performed as previously reported.25Chopra M Dharmarajan AM Meiss G Schrenk D Inhibition of UV-C light-induced apoptosis in liver cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin.Toxicol Sci. 2009; 111: 49-63Crossref PubMed Scopus (39) Google Scholar HUVECs were harvested in incubation medium and spun down at 500g for 10 minutes at 4°C, resuspended in PBS, and spun down again using the same conditions. For isolation of nuclei, cell pellets were resuspended in 500 μl of nuclei isolation buffer (10 mmol/L PIPES, 10 mmol/L KCl, 2 mmol/L MgCl2, 1 mmol/L DTT, 0.1% protease-inhibitor cocktail, pH 7.4). Cells were homogenized using a glass potter, layered over 500 μl of 30% sucrose in nuclei isolation buffer, and centrifuged for 10 minutes at 800g and 4°C. The pellet was washed with nuclei isolation buffer and nuclei were lysed with nuclei isolation buffer containing 0.1% SDS for 30 minutes on ice. Cellular debris was spun down and the supernatant stored at −80°C. C-Jun antibody was used at a dilution of 1:1000 in TBST (Cell Signaling) and HRP-labeled secondary antibody at a dilution of 1:10000 in TBST (Pierce). The blots were developed as above, and protein content was quantified using the Bradford assay. EA.hy926 cells were grown overnight (12 hours) in tissue culture dishes to attain 60% confluence, then washed once with PBS and media changed to that supplemented with different concentrations of sFRP4 (0 or 125 pg/ml). The cells were incubated for 4 hours at 37°C/5%CO2, then the medium was aspirated and cells were washed with PBS (2 times). Cells were incubated in PBS supplemented with 10 μmol/L Amplex Red in the presence of 0.5U HRP for 15 minutes at 37°C/5%CO2. The cells were scraped and centrifuged at 2000g at 4°C and the supernatant was collected. Fluorometric readings using a Cary eclipse fluorometer (Varian, CA) were recorded at 563-nm excitation and 587-nm emission wavelength; the slit width was kept at 5 nm. EA.hy926 cells were grown overnight (12 hours) in 12-well plates coated in collagen type 2 (1 × 106 cells per well). The next morning cells were washed with PBS and the media changed to medium supplemented with sFRP4 (0 or 125 pg/ml). Cells were incubated for 4 hours at 37°C/5%CO2, following which 1 mg/ml nitroblue tetrazolium was added to the media and cells incubated for a further 2 hours. After incubation the medium was aspirated and cells were washed with PBS (2 times). Formazan crystals formed by the action of superoxides on nitroblue tetrazolium were dissolved by adding 200 μl of DMSO and the optical density was measured at 540 nm using a spectrophotometer (Varian Cary 4000 uv-vis photometer, Varian, CA). EA.hy926 cells were grown overnight (12 hours) in tissue culture plates coated in collagen type 2 to attain 80% confluence, and the following morning cells were washed with PBS and the media changed to medium supplemented with sFRP4 (0 or 125 pg/ml). Cells were incubated for 4 hours in sFRP4 supplemented medium at 37°C/5%CO2. After incubation, cells were washed 2 times with PBS, scraped in ice cold PBS (500 μl/35 mm dish), and centrifuged to form a pellet at 2000g/4°C. Supernatant was discarded and the pellet was resuspended in 200 μl 50 mmol/L Tris-HCl buffer (pH 7.4). Cells were homogenized on ice using a Dounce homogenizer at 50 strokes. The sample was prepared by diluting lysate in 2 ml 0.1 mol/L PBS buffer (pH 7.4) and 0.1% H2O2 was added, after which the optical density was measured at 15-second intervals up to 3 minutes. The decrease in optical density corresponds to the breakdown of hydrogen peroxide by catalase activity. The activity of the enzyme was plotted as a function of time and expressed as the amount of H2O2 consumed/min/mg of protein. EA.hy926 cells were grown to confluence in 24-well plates as described in the wound healing assay. A wound was created as per the wound healing assay and then incubated with either superoxide dismutase (SOD) or nothing to determine whether SOD per se had any impact on wound healing. Next, cells were again grown to confluence and subjected to wounding followed by incubation with either sFRP4 (125 pg/ml) or sFRP4 (125 pg/ml) in combination with SOD (150 SI units). Wound healing was measured for the next 4 hours by taking images of the wound at 0 and 4 hours of incubation. Fourth day incubated chicken eggs were collected from the Poultry Research Station, Nandanam, Chennai. In a modification of the chicken chorioallantoic membrane assay,26Auerbach R Lewis R Shinners B Kubai L Akhtar N Angiogenesis assays: a critical overview.Clin Chem. 2003; 49: 32-40Crossref PubMed Scopus (568) Google Scholar the eggs were broken and gently plated on a cellophane bed in Petri dishes under sterile conditions. Sterile filter paper disks soaked in sFRP4 (0 or 125 pg/ml) were then placed on the egg yolks and incubated for another 12 hours at 37°C. Images were taken using a Nikon digital camera with a stereo microscope at 0, 6, and 12 hours of incubation. Quantification of angiogenesis was performed by using Image J image analysis software (Release α 4.0 3.2). Ten to 20 mg of sterile absorbent cotton plugs were implanted in the peritoneal cavity of Wistar rats with or without sFRP4 (125 pg/ml). After 8 days the animals were sacrificed and the cotton plug granulomas were extracted to measure the hemoglobin content. A polyether-polyurethane sponge (0.5 cm2) was soaked in Matrigel that had been thawed at 4°C and the sponge was implanted in the peritoneal cavity of Wistar rats with or without sFRP4 (125 pg/ml). The animals were sacrificed after eight days, and the resulting granulomas were removed to measure the hemoglobin levels. Matrigel (20 mg/ml) was injected into the peritoneal cavity of Wistar rats with or without sFRP4 (125 pg/ml). The animals were sacrificed after 8 days to extract the matrigel pellet. The hemoglobin content of the pellet was measured spectrophotometrically. Images of the implants were converted to gray scale and adjusted to suitable brightness and contrast using Adobe Photoshop 7.0 to perform automated image analysis using AngioQuant.27Niemisto A Dunmire V Yli-Harja O Zhang W Shmulevich I Robust quantification of in vitro angiogenesis through image analysis.IEEE Trans Med Imaging. 2005; 24: 549-553Crossref PubMed Scopus (152) Google Scholar Next, the images were processed with AngioQuant software as directed by the software tools. The number of junctions (which reflects the number of vessels formed in the implants) was taken as an index of angiogenesis. BALB/c nude mice were injected subcutaneously with 5 × 106 SKOV-3 cells, a human cell line derived from ovarian serous cystadenocarcinoma. On day 5, when tumors had grown to 70 to 100 mm3, 100 μg of Avastin or sFRP4 in 100 μl PBS, or 100 μl PBS alone were injected intraperitoneally twice weekly for 3 weeks. On day 43 the tumors were excised under general anesthesia and immersed in a solution of 4% paraformaldehyde in phosphate buffer (pH 7.2) and processed for paraffin embedding. Five-μm sections were made for routine hematoxylin and eosin staining followed by microscopic examination and photography. Cryostat sections of frozen tissue were cut at 6 μm, placed on Super Frost Plus slides, and stored at −20°C until use. Immediately before commencing immunostaining, the sections were washed in PBS buffer for 2 minutes before incubation with 3% H2O2 (in methanol) for 25 minutes at room temperature to block endogenous peroxide activity before 3% bovine serum albumin in PBS for 60 minutes at room temperature to block nonspecific binding. Sections were stained using Mec 13.3 antibody (rat anti-mouse CD-31) at a 1:100 dilution (BD Pharmingen, San Diego, CA) overnight at 4°C and then washed in PBS buffer (2 × 2 minutes) at room temperature. The secondary biotinylated antibody (anti-rat) was applied at a 1:500 dilution (Universal secondary, Vector Laboratories Inc., Burlingame, CA) for 90 minutes at room temperature, followed by 2 × 2 minute washes in PBS. The ABC kit (Vectastain Elite ABC, Vector Laboratories Inc., Burlingame, CA) was added for 60 minutes, again followed by 2 × 2 minute PBS washes. Diaminobenzidine (Sigma, Australia) was used as a chromogen to visualize the antibody–antigen complex. Sections were counterstained in Harris's hematoxylin for 45 seconds, dehydrated, cleared, and mounted in DPX. Slides were examined using an Olympus BX50 with MicroFire (Optronics) microscope connected to a Micro Brightfield MBF camera and using Stereo Investigator software; endothelial cells were counted at ×40 magnification by random sampling of 8 sites, each site being 150 μm × 150 μm in size. Counts were performed for PBS, sFRP4, and Avastin treatment groups. Cell death was localized in tissue by TUNEL analysis.28Berg MN Dharmarajan AM Waddell BJ Glucocorticoids and progesterone prevent apoptosis in the lactating rat mammary gland.Endocrinology. 2002; 143: 222-227Crossref PubMed Scopus (29) Google Scholar Paraffin sections were cut (4 μm) using a microtome and then dewaxed by two washes in toluene (5 minutes). Sections were hydrated through a graded series of ethanol and PBS and then incubated with proteinase K (20 μg/ml in PBS) for 30 minutes at 37°C. Thereafter an in situ apoptosis kit (Apoptosis Detection kit, Chemicon International, Temecula, CA) was used for nick-end labeling according to the manufacturer's protocol. Nuclei with DNA cleavage were visualized with DAB (3,3′-diaminobenzidine tetrahydrochloride), and sections were counterstained with methyl green. Postweaning mammary gland was used as a positive control. Labeling was visualized indirectly with peroxidase-labeled antidigoxigenin antibody. Sections that were near the maximal diameter of the tumor were selected for investigation. Slides were examined using an Olympus BX50 with MicroFire (Optronics) microscope connected to a Micro Brightfield MBF camera and, using Stereo Investigator software, both normal and apoptotic endothelial cells were counted at ×40 magnification by random sampling of 8 sites, each site being 150 μm × 150 μm in size. Counts were performed for PBS, sFRP4, and Avastin treatment groups. Initiation of apoptosis was determined using the JC-1 technique.29White L Dharmarajan A Charles A Caspase-14: a new player in cytotrophoblast differentiation.Reprod Biomed Online. 2007; 14: 300-307Abstract Full Text PDF PubMed Scopus (15) Google Scholar At the onset of apoptosis the mitochondrial membrane is rapidly depolarized. When the mitochondrial membrane is polarized the JC-1 dye (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-carbocyanine iodide) aggregates and fluoresces red. On depolarization, JC-1 forms a green fluorescent monomer, so the ratio of aggregated to monomeric JC-1 gives a quantitative representation of the extent of mitochondrial membrane permeability. Apoptotic cells primarily demonstrate green fluorescence, whereas healthy cell fluoresce red/green. Briefly, cells were grown at 1 × 105 c/ml in 96-well plates for 24 hours. After either 1 or 3 hours of 1 μmol/L STS treatment, media was removed and 50 μl of 2.5 mmol/L JC-1 (Molecular Probes T-3168) dye diluted 1:75 in Hams F12-K was
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