Fig. 3. HNSCC-associated caspase-8 mutants fail to mediate TRAIL-induced apoptosis with similar potency to WT caspase-8.

A HeLa-CASP8 KO cells engineered for DOX-inducible expression of WT caspase-8, MT caspase-8 proteins, or LacZ were pre-treated in quadruplicate with DOX (1 μg/mL), followed by treatment with DOX plus varying concentrations of TRAIL for an additional 12 h before the performance of crystal violet (upper panel) or MTT (lower panel) assays. Columns represent the means from quadruplicate wells; error bars represent the SEM. One-way ANOVA was used to compare multiple groups. Experiments were performed three times with similar results. B Cells were incubated for 16 h in the absence of treatment, for 16 h with DOX (1 μg/mL) alone, or for 4 h with DOX followed by 12 h with DOX plus TRAIL (200 ng/mL). Flow cytometry for annexin V staining was performed to evaluate the induction of apoptosis. Columns represent the average percentage of annexin V-positive cells from two independent experiments; error bars represent the SEM. Student’s t test was performed to compare differences between groups with and without TRAIL treatment. C Parental PE/CA-PJ49 cells or PE/CA-PJ49-CASP8 KO cells were treated for 24 h with the indicated concentrations of TRAIL, followed by the performance of MTT assays. Columns represent the means from eight wells; error bars represent SEM. Student’s t test was performed to compare groups without TRAIL treatment and with TRAIL at 25 ng/mL or 200 ng/mL. D PE/CA-PJ49-CASP8 KO cells engineered for DOX-inducible expression of WT caspase-8 or MT caspase-8 (D303G) were treated for 24 h with DOX plus vehicle (Veh) or DOX plus TRAIL (200 ng/mL), followed by the performance of MTT assays. Columns represent the means from quadruplicate wells; error bars represent the SEM.