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. 2022 Mar 23;298(5):101850. doi: 10.1016/j.jbc.2022.101850

Figure 6.

Figure 6

GSDME and caspase-3 are necessary for EV71-mediated pyroptosis.A, generation of Gsdme−/− HeLa cells by CRISPR–CRISPR-associated protein 9 (Cas9)–mediated genome editing. The right panel shows a related portion of the GSDME genomic structure. The single-guide RNA (sgRNA) sequence is shown in red. One screen-positive clone of Gsdme−/− was detected by Western blotting. B, generation of caspase-3 and GSDME double-KO (DKO) HeLa cells. DKO cells were established using as sgRNA of caspase on the basis of Gsdme−/− HeLa cells. The sgRNA sequence of caspase-3 is shown in red. Expression of caspase-3 and GSDME was detected in the DKO cells by Western blotting. C and D, Gsdme−/−, caspase 3−/−, and DKO cells were mock infected or infected with EV71 at an MOI of 2. At 24 h after infection, LDH release–based cell death (C) and ATP cell viability (D) were detected. E and F, EV71-induced pyroptosis and viral replication in WT, caspase 3−/−, Gsdme−/−, and DKO cells. The four cell lines were infected with EV71 at an MOI of 2. After 24 h, phase-contrast imaging was performed. Images were obtained by microscopy. The scale bar represents 50 μm. Cell lysates were prepared for Western blotting. All data are mean values ± SD. Experiments were performed as triplicates and repeated at least twice. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Data are representative of three independent experiments. EV71, enterovirus 71; GSDME, gasdermin E; MOI, multiplicity of infection.