Fig. 7.

PANX1 promotes gasdermins and MLKL-independent cell lysis during TNF-induced cell death. A: representative images of WT, ABCDEM-6KO, PANX1-deficient, and PANX1-deficient in 6KO HeLa-pTet-nSREBP2 cell treated with 50 ng/ml TNF and 10 μg/ml CHX for 24 h. Scale bar, 25 μm. B: LDH release test. Data are presented as means ± SD. Statistical analyses, unpaired two-tailed Student’s t test. C: PANX1 knockout in ABCDEM-6KO HeLa-pTet-nSREBP2 cells were transfected with wild-type or mutated PANX1 (D379A). All cells were treated with TNF and CHX for 24 h. LDH tests were applied to analyze lytic cell death. Data are presented as means ± SD. Statistical analyses, unpaired two-tailed Student’s t test. D: cell viability test. HeLa cells were plated in 10% FBS medium or sterol depletion medium (5% lipoprotein-deficient serum, 1 μM lovastatin, 10 μM mevalonate) for 16 h, then treated with indicated concentrations of TNF (starting from 30 ng/ml, with 3-fold serial dilutions) and 10 μg/ml CHX for 8 h. Cell viabilities were determined by CCK8 and normalized to CHX only groups. Nonlinear regression was performed in Prism and EC₅₀ was calculated. E: LDH release test. HeLa-pTet-nSREBP2 cells were plated with 0/0.03 μg/ml dox for 8 h, then treated with indicated TNF and 10 μg/ml CHX for 8 h. F: representative images of WT, ABCDEM-6KO, PANX1-deficient, and PANX1-deficient in 6KO cell treated with TNF and AZD5582 for 24 h, cells were preloaded with PI for 15 min and imaged at 63× magnification. Scale bar, 25 μm. G: LDH release test. H: PANX1-deficient in ABCDEM-6KO HeLa-pTet-nSREBP2 cells were transfected with wild-type or mutated PANX1 (D379A). All cells were treated with 50 ng/ml TNF and 5 μM AZD5582 for 24 h. LDH tests were applied to analyze lytic cell death. Data are presented as means ± SD. Statistical analyses, unpaired two-tailed Student’s t test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. ns, not significant.