Journal: Cell host & microbe
Article Title: Lipid Peroxidation Drives Gasdermin D-mediated Pyroptosis in Lethal Polymicrobial Sepsis
doi: 10.1016/j.chom.2018.05.009
Figure Lengend Snippet: (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).
Article Snippet: RNAi and gene transfection Mouse PLCG1-shRNA (#TRCN0000024974 and #TRCN0000024976), human PLCG1-shRNA (#TRCN0000218478), and control-shRNA (#SHC001) were obtained from Sigma (St. Louis, MO, USA).
Techniques: Transfection, Expressing, Western Blot, shRNA, Electroporation, Infection
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