Fig. 5: Effect of GZMA or NE cleavage on the pyroptotic activity of GSDMB isoforms.
A Lactate dehydrogenase (LDH) assay in transiently transfected (48 h) HEK293T cells expressing 1–275 cytotoxic fragment and the corresponding individual mutants of GZMA-cleavage site 1–275K229A and 1-275K244A. Values represent means ± SEMs of 6 independent experiments. Differences between cytotoxic 1–275 and other constructs were tested by two-tailed unpaired t-test: ****p < 0,0001; ns, not significant. B HEK293T cells expressing GSDMB isoforms [1,2,3,4] were co-cultured with NK-92 cells. Immunoblotting shows the cleavage of GSDMB by GZMA using anti-GSDMB-NT (Sigma, HPA023925) and anti-GSDMB-CT antibody Ab-GB [39]. C Lactate dehydrogenase (LDH) assay after 16 h of NK-92 co-culture and (D) Mitochondrial damage of HEK293T analyzed by mitoSOX using flow cytometry. NK-92 cells were labelled with CD56 antibody. LDH and MitoSOX values represent means ± SEMs of four independent experiments. Differences between control condition (HEK293T empty vector co-culture with NK-92) and each condition was tested by two-tailed unpaired t-test: *p < 0,05, **p < 0,01 and ***p < 0,001. E Immunoblotting analysis of GSDMB cleavage by human neutrophil elastase (rhNE). HEK-293T cell lysates containing GSDMB isoforms [1,2,3,4] were incubated with rhNE (68 nM) at 37 °C for 30 min (F) and in presence of indicated amount of BAY-678 inhibitor. Immunoblot probes for GSDMB cleavage detection: anti-GSDMB-NT (Sigma, HPA023925) and anti-GSDMB-CT antibody Ab-GB [39]. GAPDH was used as a loading control. G Lactate dehydrogenase (LDH) assay after 48 h of co-transfection of hNE vector and GSDMB constructions. Values represent means ± SEMs of six independent experiments. Differences between hNE treated and untreated conditions were tested by two-tailed unpaired t-test: *p < 0,05 and **p < 0,01. H HEK293T were transfected with hNE vector and subsequently with GSDMB constructs. GSDMB-NT constructs were cleaved by hNE, and this produce a decrease in the levels of GDSMB-NT fragments. Fragments were detected by anti-Myc flag located at the C-terminal region of GSDMB. HSP90 was used as a loading control.
