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. 2021 Dec 29;298(2):101553. doi: 10.1016/j.jbc.2021.101553

Figure 7.

Figure 7

Antioxidant therapy with NAC represses activation of GSDME by caspase-3 to prevent rupture of the plasma membrane of atRAL-loaded 661W photoreceptor cells.A, rupture of the plasma membrane was assessed by LDH release assay. 661W photoreceptor cells were incubated with 5 μM of atRAL for 6 h in the presence or the absence of 2 mM of NAC. Note that cells were pretreated with NAC for 2 h. Cells treated with NAC or DMSO alone served as controls. F0.05 (1,20) = 103.7, p < 0.0001. B and C, immunoblot analysis of HMGB1 in cell lysates or culture supernatants from 661W photoreceptor cells exposed to 5 μM of atRAL for 6 h with 2 mM of NAC. Note that cells were pretreated with NAC for 2 h. Protein levels of sup. HMGB1 were normalized to those of GAPDH and expressed as fold changes compared with DMSO-treated WT controls. F0.05 (1,8) = 399.8, p < 0.0001. D, 661W photoreceptor cells were preincubated with 2 mM of NAC for 2 h and then treated with 5 μM of atRAL for 6 h. Cell lysates were collected and subjected to Western blot analysis of procaspase-3, cleaved caspase-3, GSDME-FL, and GSDME-N. E, protein levels of cleaved caspase-3 (F0.05 (1,8) = 69.28, p < 0.0001) and GSDME-N (F0.05 (1,8) = 223.5, p < 0.0001) were normalized to those of GAPDH and expressed as fold changes compared with DMSO-treated WT controls. Statistical analyses in A, C, and E were performed by using two-way ANOVA with Tukey's post-test. atRAL, all-trans-retinal; DMSO, dimethyl sulfoxide; GSDME, gasdermin E; GSDME-FL, full-length GSDME; GSDME-N, N-terminal fragment of GSDME; HMGB1, high-mobility group box 1; LDH, lacate dehydrogenase; NAC, N-acetyl-l-cysteine; sup., supernatant.