Fig. 3: Procaspase-8 mutations at the putative methylation sites inhibit CD95L-induced caspase activity and apoptosis.

A, B HeLa-CD95-C8-KO cells, which were transfected with pcDNA3 (vector), procaspase-8a-R233H-pcDNA3 (R233H), procaspase-8a-R435Q-pcDNA3 (R435Q) and procaspase-8a-WT-pcDNA3 (WT) or non-transfected (KO), were treated with the indicated amounts of CD95L for the indicated time intervals. Caspase-8 and -3 activities were determined by Caspase-Glo^®8 Assay (A) and Caspase-Glo^®3/7 Assay (B), respectively. Western Blot controls of transfection efficiency are shown in the lower part of the panels. C The HeLa-CD95-C8-KO cells were transfected as described before. This was followed by treatment with 500 ng/mL CD95L (+) for one hour and Western Blot analysis with the indicated antibodies were performed. One representative Western Blot out of three independent experiments is shown. D–F The HeLa-CD95-C8-KO cells were transfected as described before. Cells were treated with the indicated amounts of CD95L for indicated time intervals. Representative Western Blot analysis of transfection efficiency is shown in the lower part of the panels. D Cell viability was captured by measuring ATP levels using CellTiter-Glo^® 2.0 Cell Viability Assay. E Metabolic activity was captured by measuring metabolic level using RealTime-Glo™ MT cell Viability Assay. F LDH release was measured by LDH-Glo^® Cytotoxicity Assay. G, H HeLa-CD95-C8-KO cells were transfected as described above. After 24 hours, the cells were treated with indicated amounts of CD95L for 6 hours. Cells were stained by Annexin-V-FITC (An) and Propidium Iodide (PI) and then analysed by Imaging Flow Cytometry. G The amounts of viable cells (gated negative cells) after CD95L treatment are shown. H The amounts of An+ single-positive and An + /PI+ double positive cells after CD95L treatment are shown. I Representative images of viable (negative), An+ single-positive, An + /PI +  positive (double-positive) cells. J The representative Western Blot analysis of transfection efficiency is shown. A, B, D–H Mean and standard deviation are shown for three independent experiments. Statistical analysis was carried out by ordinary ONE-WAY ANOVA with subsequent Tukey-test (** significant; p < 0.01; *** significant; p < 0.001; **** significant; p < 0.0001). s.e. short exposure, l.e. long exposure, Bf bright field, An, Annexin V-FITC, PI Propidium Iodide.