Figure 1.
Impairment of FasL-induced cell death in caspase-8 and -10-doubly deficient Jurkat cells.
A, 20 µg total protein extracts from parental (A3) and caspase-8 deficient (I9-2a, b, d and e) Jurkat cell lines were subjected to SDS-PAGE, and Western blotted with anti-caspase-8, -10, -2, -9, -3, -7, anti-FADD, anti-RIP or anti-β-actin antibodies. B, Jurkat cells were labelled with anti-CD95-PE antibody (thick line) or an isotype control (thin line) and analysed by flow cytometry. Percentages of cells expressing CD95 are indicated. C, Jurkat cells were incubated for 8 hours in the presence of 500 ng/mL FasL and cell viability was evaluated by MTT assay. Values are means ± SEM of three independent experiments. *p<0.05; **p<0.01, *** p<0.001 as compared to values obtained in A3 cells.
Figure 2.
Impairment of FasL-induced caspase activation and apoptosis in caspase-8 and -10-doubly deficient Jurkat cells.
A, B, A3 (white bars), I9-2d (grey bars) and I9-2e (black bars) Jurkat cells were incubated for 8 hours in the presence or absence of 500 ng/mL FasL as indicated. Caspase activities were assessed using Ac-DEVD-AMC or Ac-IETD-AMC (A). Cells were stained with annexin-V-FITC and propidium iodide and analyzed by flow cytometry. Percentages of annexin-V-positive (AnV+) are indicated (B). All data are means ± SEM of three to four independent experiments. *p<0.05; **p<0.01, *** p<0.001. C, Representative flow cytometry experiment. Low right quadrants: percentages of [AnV-FITC+ propidium iodide (PI)-] cells; Up right quadrants: percentages of [AnV-FITC+ PI+] cells; Up left quadrants: percentages of [AnV-FITC- PI+] cells.
Figure 3.
Caspase-8 and -10 can restore FasL-induced apoptosis in caspase-8 and -10-doubly deficient Jurkat cells.
A, B, I9-2e Jurkat cells were transiently transfected with plasmids encoding EGFP, EGFP-tagged wild-type caspase-8 or -10 or EGFP-tagged catalytically inactive caspase-8 mutant (Casp-8 C360S). Alternatively, cells were co-transfected with plasmids encoding EGFP and catalytically inactive caspase-10 mutant (C401S). Twelve hours post-transfection, cells were further incubated for 4 hours in the presence or absence of 500 ng/mL FasL. Cells were labelled with annexin-V-APC (AnV-APC). EGFP fluorescence was monitored on untreated cells to determine the percentage of transfected cells (A). Analysis of annexin-V binding was restricted to the EGFP-expressing cells on both untreated (None) and FasL-treated (FasL) cells (B). Percentages of fluorescent cells are indicated. Data are from a representative experiment out of three independent experiments. C, Specific cell death triggered by FasL was calculated by subtracting basal values obtained in the absence of FasL for each transfection. Data are means ± SEM of three independent experiments. *p<0.05; **p<0.01.
Figure 4.
Impairment of FasL-induced cell death in the presence of zVAD-fmk in caspase-8 and -10-doubly deficient Jurkat cells.
A, A3 cells were incubated for the indicated times with 500 ng/mL FasL in the presence (open symbols) or absence (filled symbols) of 40 µM zVAD-fmk. Caspase activities were assessed using Ac-DEVD-AMC (circles) or Ac-IETD-AMC (triangles). Data are representative of two independent experiments. B, A3 and I9-2e cells were incubated for 8 hours in the presence or absence of 500 ng/mL FasL and zVAD-fmk (40 µM) as indicated. Cells were fixed and stained with DAPI before microscopy examination. Arrows indicate cells with partial chromatin condensation. C, D, A3, I9-2d and I9-2e cells were incubated for 8 hours in the presence of zVAD-fmk (40 µM) with or without 500 ng/mL FasL as indicated. Cells were stained with Annexin-V-FITC (AnV-FITC) and propidium iodide (PI) and analysed by flow cytometry. Representative flow cytometry experiment out of four independent experiments (C). Low right quadrants: percentages of [AnV-FITC+ PI-] cells; Up right quadrants: percentages of [AnV-FITC+ PI+] cells; Up left quadrants: percentages of [AnV-FITC- PI+] cells. D, Percentages of anexin-V-positive (AnV+) cells (means ± SEM, n = 4). **p<0.01.
Figure 5.
Caspase-10 can restore FasL-induced cell death in the presence of zVAD-fmk in caspase-8 and -10-doubly deficient Jurkat cells.
A, B, I9-2e Jurkat cells were transiently transfected as described in the legend to Figure 3 and immediately incubated in the presence of 40 µM zVAD-fmk. Twelve hours post-transfection, cells were further incubated for 4 hours in the presence or absence of 500 ng/mL FasL. Cells were labelled with annexin-V-APC (AnV). EGFP fluorescence was monitored on untreated cells to determine the percentage of transfected cells (A). Annexin-V binding analysis was restricted to the EGFP-expressing cells on both untreated (None) and FasL-treated (FasL) cells (B). Percentages of fluorescent cells are indicated. Data are from a representative experiment out of three independent experiments. Of note, the present data were obtained in the same experiment depicted in Fig. 3B. C, Specific cell death triggered by FasL was calculated by subtracting basal values obtained in the absence of FasL for each transfection. Data are means ± SEM of three independent experiments. **p<0.01.
Figure 6.
zVAD-fmk does not abrogate over-expressed caspase-10-triggered cell death in HeLa cells.
A, Total protein extracts derived from wild-type Jurkat (A3) and HeLa cells were analysed by Western blot using anti-caspase-8, anti-caspase-10 or anti β-actin antibodies. B–D, HeLa cells were transfected with plasmid encoding EGFP, EGFP-tagged wild-type caspase-8 (Casp8) or -10 (Casp10). B, Cells were analysed by flow cytometry 16 hours post-transfection. Values indicated are percentages of EGFP-expressing cells. C, Immediately after transfection, 40 µM zVAD-fmk was added or not to the cell culture medium and cells were further incubated for 24 hours. During the last 8 hours, cells were incubated with or without 1 µg/mL FasL. Protein extracts were analysed by Western blot using the indicated antibodies. Cleaved PARP expression was analysed at low (up panel) and high (low panel) exposure. (*: EGFP-tagged pro-caspase-8 or 10, **: EGFP-tagged small catalytic caspase-8 or -10 subunit). The ≈26 kDa band obtained in caspase-8 and -10 expressing cells remains to be characterized. D, HeLa cells were transfected and cultured for 24 hours in the presence or absence of zVAD-fmk. Cells were labelled with annexin-V/APC. Annexin-V binding analysis was restricted to the EGFP-expressing cells. Data are means ± SEM of five independent experiments. ***p<0.001; * p<0.05.
Figure 7.
Generation of HeLa cells stably expressing EGFP-tagged wild-type caspase-10 at low level.
A, HeLa cells were transfected with plasmid encoding EGFP-tagged wild-type caspase-10 and incubated for 45 days in DMEM 10% FCS containing 0.8 mg/mL G418. The whole population of cells (Casp10) were analysed by flow cytometry and untransfected HeLa WT cells were used as negative control to determine the threshold for FL1 channel. Values are percentages of positive cells in FL1 channel. B, Scheme for selection of cells expressing (Casp10+) or not (Casp10-) EGFP-tagged caspase-10. C, Casp10+ and Casp10- cells were FACS sorted from the heterogeneous population stably expressing caspase-10 and further cultured for one month in DMEM 10% FCS containing 0.8 mg/mL G418 before analysis by flow cytometry for EGFP-tagged caspase-10 and CD95 expression. Values indicated percentages of positive cells in FL1 (up panels) and FL2 (low panels) channels. D, Wild-type Jurkat (A3), Casp10+ and Casp10- HeLa cells were analysed by Western blot using the indicated antibodies. Caspase-10 expression was analysed at low (up panel) and high (low panel) exposure. (*: EGFP-tagged pro-caspase-10).
Figure 8.
zVAD-fmk does not totally abrogate FasL-triggered apoptosis in HeLa cells expressing caspase-10 at low level.
A, B, Casp10+ and Casp10- HeLa cells were incubated in the presence or absence of 40 µM zVAD-fmk for 1 hour and further incubated for 16 hours with or without 1 µg/mL FasL as indicated. Cells were labelled with Syto13 and propidium iodide before fluorescence microscopy examination (A). Of note, less than 5% of the cells were stained by propidium iodide under all conditions indicating that FasL-induced necrosis was marginal in HeLa cells. Percentages of cell death (i.e., cells exhibiting nuclear fragmentation and/or condensation) were determined by analysing at least 500 cells for each condition. Values are means ±SEM of three independent experiments (n.s.: not significant; **p<0.01.) (B). C, Casp10+ and Casp10- HeLa cells were incubated in the presence or absence of 40 µM zVAD-fmk for 1 hour and further incubated for 8 hours with or without 1 µg/mL FasL as indicated. Western blot analysis was performed using the indicated antibodies. (*: EGFP-tagged pro-caspase-10, **: EGFP-tagged small catalytic caspase-10 subunit).