Fig. 2: PANX1 cleavage is dispensable for caspase-dependent apoptosis but essential for ATP release.

A HCT15 and HCT116 cells were treated with TNFα (50 ng/mL) and the caspase inhibitor z-VAD-fmk (10 μM) for 24 h. The cleavage of caspase-3 and PANX1 was evaluated by western blotting (n = 3). **p < 0.01 and ***p < 0.001. One-Way ANOVA test. B HCT15 and HCT116 cells were treated with TNFα (50 ng/mL) and the caspase inhibitor z-VAD-fmk (10 μM) for 24 h. Caspase-3 activity was evaluated with a caspase-3 activity kit (n = 3). *p < 0.05 and **p < 0.01. One-Way ANOVA test. C HCT15 and HCT116 cells were treated with TNFα (50 ng/mL) and the SFK inhibitor quercetin (10 μM) for 24 h. The cleavage of caspase-3 and PANX1 was evaluated by western blotting (n = 3). ***p < 0.001. One-Way ANOVA test. D HCT15 and HCT116 cells were infected with lentivirus carrying shRNA targeting PANX1 and selected with puromycin for 3 days. The knockdown efficacy of lentivirus carrying shRNA targeting PANX1 was evaluated by western blotting (n = 3). ***p < 0.001. Unpaired t-test. E HCT15^shNC and HCT15^shPANX1 cells were treated with TNFα (50 ng/mL) for 24 h. The cleavage of caspase-3 was evaluated by western blotting (n = 3). Similar experiments were carried out in HCT116 cells. **p < 0.01. One-Way ANOVA test. F HCT15^shNC and HCT15^shPANX1 cells were treated with TNFα (50 ng/mL) for 24 h. Caspase-3 activity was evaluated with a caspase-3 activity kit (n = 3). Similar experiments were carried out in HCT116 cells. *p < 0.05. One-Way ANOVA test. G HCT116^shNC and HCT116^shPANX1 cells were treated with TNFα (50 ng/mL) for 24 h. Apoptotic cells were examined by flow cytometry (n = 3). *p < 0.05. One-Way ANOVA test. H HCT116^shNC and HCT116^shPANX1 cells were treated with TNFα (50 ng/mL) for 6 h. The intracellular ATP content was examined by flow cytometry (n = 3). *p < 0.05. One-Way ANOVA test.