Fig. 2

Inhibition of proteasome induces mitotic pyroptosis via GSDME. a, b Proteasome inhibition induced morphology of pyroptosis. Five random fields in each well were captured and then subjected to analysis for the rate of cells with pyroptosis morphology. One of the five fields is shown as representative image for each group. Red arrows indicate the pyroptotic cells with large ballooning bubbles. The proportion of pyroptotic cells was calculated (right panel). Scale bar, 20 μm. c, d Proteasome inhibition stimulated LDH release. e, f GSDME silencing attenuated the proteasome inhibition-induced increase of pyroptotic cells. g, h GSDME knockdown abrogated proteasome inhibition-induced LDH release. For e–h, SNU449 cells were transfected with NC or siRNA targeting the indicated gasdermins (siGSDMs) for 24 h, then treated with 15 nM BTZ for another 48 h (e, g), or cells were co-transfected with siGSDMs and siPSMC5-1/2 for 72 h (f, h) before phase-contrast imaging (e, f) or LDH release assay (g, h). i, j Proteasome inhibition induced translocation of GSDME to the plasma membrane of multi-polar mitotic cell. White arrows indicate the clusterization of GSDME on cell membrane. Scale bar, 2.5 μm. k, l Proteasome inhibition induced the cleavage of caspase-3 and GSDME. SNU449 cells were treated with 15 nM BTZ for 48 h (a, c, i, k), or transfected with the indicated RNA duplexes for 72 h (b, d, j, l) before phase-contrast imaging (a, b), LDH detection (c, d), immunofluorescent staining for GSDME (Red), α-tubulin (TUBA, green) and chromosomes (DAPI, blue) (i, j), or immunoblotting (k, l). #, unspecific band. m Silencing caspase-3 but not caspase-1 blocked the BTZ-induced GSDME cleavage. n Silencing cGAS but not CHOP or IκBα attenuated the BTZ-induced cleavage of caspase-3 and GSDME. For m, n, SNU449 cells were transfected with NC or the indicated siRNA for 24 h, then treated with 15 nM BTZ for another 48 h before immunoblotting. o Ectopic expression of BCL-xL attenuated the BTZ-induced cleavage of caspase-3 and GSDME. SNU449-BCL-xL and its control line SNU449-Ctrl were treated with vehicle or 15 nM BTZ for 48 h before immunoblotting. Red arrows indicate the target band. p BTZ-induced cleavage of GSDME was enhanced by nocodazole but was inhibited by CDK1 inhibitor RO-3306. SNU449 cells were pretreated with vehicle, 50 ng/mL nocodazole or 10 μM RO-3306 for 6 h, followed by treatment with vehicle or 15 nM BTZ for another 48 h before immunoblotting. Error bars: SEM from at least three independent experiments. Student’s t test (a and c) and one-way ANOVA (b and d–h) were used. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns not significant