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

Figure 5.

Figure 5

EV71-mediated cleavage of GSDME is dependent on activation of caspase-3.A, primary sequences of amino acids 262 to 274 in GSDME. B, 293T cells were transfected with plasmids that encoded WT GSDME or D270A mutant GSDME. At 24 h after transfection, cells were mock infected or infected with EV71 at increasing doses for 24 h. Cell lysates were analyzed by Western blotting. C, Casp3−/− HeLa cells were generated by CRISPR–CRISPR-associated protein 9 (Cas9)–mediated genome editing. The upper of (C) shows a related portion of the caspase-3 genomic structure. The single-guide RNA sequence is shown in red, which resided in exon 5 of caspase-3. Sequences of the targeted region and KO are shown. The lower of (C) shows expression of caspase-3 in WT and KO cells. D, WT and caspase-3-KO cells were infected with EV71. At the indicated times, cells were collected and lysates were analyzed by Western blotting. Data are representative of three independent experiments. E, the WT HeLa and other clones of Casp3−/− HeLa cells were infected with EV71. At 24 h, cells were collected and lysates were analyzed by Western blotting. F, the plasmid that expresses caspase-3 was transfected into the Casp3−/− HeLa cells. After 24 h, cells were infected with EV71. At 24 h, the GSDME cleavage and caspase-3 expression were detected by Western blotting. EV71, enterovirus 71; GSDME, gasdermin E.