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

Fig. 4.

Fig. 4

nSREBP2 triggers cell death in a caspase-dependent manner. A: effects of different cell death inhibitors on dox-induced cell death. HeLa-pTet-nSREBP2 cells were treated with or without 0.3 μg/ml dox and inhibitors for 24 h. Cell viability was analyzed by CCK8. z-VAD (z-VAD-FMK), 50 μM; Fer-1 (ferrostatin-1), 38 μM; Nec-1s (necrostatin-1s), 30 μM. Data are presented as means ± SD. Statistical analyses, unpaired two-tailed Student’s t test. B: representative images of HeLa-pTet-nSREBP2 cells treated with or without z-VAD. All cells were treated with or without 50 μM z-VAD and with 0.3 μg/ml dox for 24 h, then imaged at 40× magnification at 5 min intervals for 12 h. Scale bar, 25 μm. C: effects of dox induction on intracellular processing of caspase-9 and caspase-10. D: effects of caspase-9/10 on dox-induced cell death. WT and caspase-9/10 double deficient HeLa-pTet-nSREBP2 cells were treated with or without 0.3 μg/ml dox for 36 h, and lytic cell death was analyzed by LDH. Data are presented as means ± SD. Statistical analyses, unpaired two-tailed Student’s t test. E: immunoblotting of caspase-3 and caspase-7 expression on dox induction in WT, caspase-3 deficient and caspase-3/7 double-deficient HeLa-pTet-nSREBP2 cells. F: Crystal violet staining. G: quantification of cell viability in F. H: effects of caspase-3 and 7 on dox-induced lytic cell death were analyzed by LDH tests. Data are presented as means ± SD. Statistical analyses, unpaired two-tailed Student’s t test. I: representative images of the effect of caspase-7 overexpression on caspase-3/7 double-deficient HeLa-pTet-nSREBP2 cells. The caspase-3/7 double-deficient HeLa-pTet-nSREBP2 cells were pre-infected with or without caspase-7 expression lentivirus, all cells were treated with 0.3 μg/ml dox for 24 h, then imaged at 40× magnification at 5 min intervals for 12 h. All cells were preloaded with 10 μg/ml PI for 15 min. Real-time videos are included in supplemental Video S4. Scale bar, 25 μm. J: the effect of caspase-7 overexpression on caspase-3/7 double-deficient HeLa-pTet-nSREBP2 cells. The caspase-3/7 double-deficient HeLa-pTet-nSREBP2 cells were pre-infected with or without caspase-7 expression lentivirus, all cells were treated with or without 0.3 μg/ml dox for 36 h. Lytic cell death was analyzed by LDH test. K: WT and caspase-3/7-deficient HeLa-pTet-nSREBP2 cells were infected with or without wild-type and mutated caspase-7 (C186A). Cell viability was analyzed by CCK8. ns, not significant, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.