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. 2024 Jun 14;65(7):100579. doi: 10.1016/j.jlr.2024.100579

Fig. 6.

Fig. 6

Requirement of PANX1 as the mediator for nSREBP2-induced membrane rupture. A: scheme depicting the screening of PANX1 as the mediator for nSREBP2-induced cell death. The Caspase-3-deficient HeLa-pTet-nSREBP2 cells were used for CRISPR/cas9 screening. B: genes were ranked according to the enrichment of sgRNA counts. The dotted line represents a fold change of 2. C: the list of top 10 genes from B. D: The nSREBP2 expression-induced LDH release in WT and APAF1-deficient HeLa-pTet-nSREBP2 cells. WT and APAF1-deficient HeLa-pTet-nSREBP2 cells were treated with 0/0.3 μg/ml dox for 36 h. E: immunoblotting assay. All cells were treated with 0/0.3 μg/ml dox for 16 h. F: the nSREBP2 expression-induced LDH release in WT and PANX1-deficient HeLa-pTet-nSREBP2 cells. Data are presented as means ± SD. Statistical analyses, unpaired two-tailed Student’s t test. G: representative confocal images of PI-stained WT and PANX1-deficient HeLa-pTet-nSREBP2 cells. All cells were induced by 0/0.3 μg/ml dox for 36 h, then imaged at 63× magnification with 10 μg/ml PI. Scale bar, 25 μm. H: flow cytometry of PI-stained WT and PANX1-deficient HeLa-pTet-nSREBP2 cells. I: effect of CBX on dox-induced lytic cell death was analyzed by LDH test. WT and ABCDE-5KO HeLa-pTet-nSREBP2 cells were treated with 0/0.3 μg/ml dox and 50 μM CBX for 36 h. J, Effects of overexpression of PANX1 on cell death. Full length (FL) or truncated (ΔCTT) PANX1 in HeLa-pTet-nSREBP2 cells were induced for 24 h by 2 μg/ml dox. Cell viability was analyzed by cell counting kit-8. K, LDH release test on the HeLa cells expressing full length (FL) or truncated (ΔCTT) PANX1. Cells were treated with or without CBX and dox for 36 h. l, Representative images of truncated (ΔCTT) PANX1-expressing HeLa cells under dox induction. Cells were treated with 2 μg/ml dox for 24 h, then preloaded with 10 μg/ml PI and imaged at 63× magnification at 5 min intervals for 12 h. Real-time videos are included in supplemental Video S6. Scale bar, 25 μm. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001. ND, not detected.