(A) WT and Gpx4−/− BMDMs were
pretreated with or without vitamin E (100 μM) for three hours, and then
transfected with GSDMD-N. Protein expression was assayed using western blot. (B)
Analysis of GSDMD-N-mediated cytotoxicity, DAG, IP3, and calcium production at
24 hours in WT and Gpx4−/− BMDMs in
the absence or presence of Plcg1-shRNA, U73122 (10 μM),
or BAPTA-AM (10 μM) (n=3 wells/group; data expressed as means
± SD, *, P<0.05 versus GSDMD-N group, t
test). (C) Analysis of GSDMD-N-mediated cytotoxicity at 24 hours in THP1, HL-60,
and HeLa cell lines (n=3 wells/group; data expressed as means ±
SD, *, P<0.05, t test). (D) WT
BMDMs were pretreated with indicated lipid components (20 μM) for three
hours, and then transfected with GSDMD-N for 24 hours. Protein expression was
assayed using western blot. (E) Analysis of GSDMD-N/PI4P- or
GSDMD-N/PI(4,5)P2-mediated cytotoxicity at 24 hours in BMDMs in the absence or
presence of Plcg1-shRNA, U73122 (10 μM), or BAPTA-AM
(10 μM) (n=3 wells/group; data expressed as means ± SD,
*, P<0.05 versus control group, t test). (F)
Analysis of LPS electroporation- or E. coli (MOI=25)
infection-mediated cytotoxicity at 16 hours in BMDMs in the absence or presence
of Plcg1-shRNA or U73122 (10 μM) (n=3
wells/group; data expressed as means ± SD, *,
P<0.05 versus control group, t test). (G) Analysis of LPS
electroporation- or E. coli (MOI=25) infection-mediated
protein expression at 16 hours in BMDMs in the absence or presence of
Plcg1-shRNA or U73122 (10 μM).