Figure 3.

After GSDME expression was upregulated by decitabine, pyroptosis was increased. (a and b) The toxicity of decitabine to Y79 and WERI-RB-1 cells was detected by CCK-8 assay at different concentrations of decitabine. (c and d) The mRNA expression of GSDME was detected using RT-PCR after treatment with different concentrations of decitabine for 3 days (the cells were centrifuged, and decitabine was replaced every day). (e and f) The protein level of GSDME in cells was quantitatively analysed by western blot. (g and h) The protein level of GSDME in cells was detected using immunofluorescence. (i and j) CCK-8 assay of Y79 and WERI-RB-1 cell viability after treatment with carboplatin or decitabine plus carboplatin. (k) The death morphologies of the three cell lines were observed using an Olympus microscope. Y79 and WERI-RB-1 cells were treated with decitabine plus carboplatin; ARPE-19 cells were treated with decitabine; black arrows, apoptotic cells; white arrows, pyroptotic cells. (l–p) GSDME-N and its pathway proteins were detected by WB. (q and r) Flow cytometry showed the ratio of FITC monostaining to FITC and PI double-positive staining after treatment with decitabine plus carboplatin, and quantitative maps were drawn for carboplatin-treated cells after 24 h. (s) LDH in the cell supernatant was detected by ELISA. Each experiment was repeated at least three times. Scale bar, 100 μm.