Fig 1: PDK1-RSK signal is required for Caspase-8 blockade during SopF-mediated S. Typhimurium infection. (a, b) Streptomycin-pretreated C57BL/6 mice were orally infected with 5 × 107 CFU of STM-WT, STM-?sopF or STM-?sopF/psopF. IECs were isolated from ceca of mice at 12 hpi, 48 hpi and 120 hpi. (c) 293 T cells were transfected with an empty vector or pcDNA3.1-SopF-6*His-T2A-GFP for 48 h. (d) Caco-2 cells were infected with STM-?sopF carrying an empty vector or a vector expressing SopF: His at an MOI of 100 for 2 hpi and 24 hpi. (c and d) His-tagged SopF were immunoprecipitated from cell lysates and assessed for their ability to bind endogenous PDK1. (e-h) After being pre-treated with mock (DMSO, 1 µL/mL), PDK1 inhibitor (AR-12, 1 µM) or RSK inhibitor (BI-D1870, 10 µM) for 1 h, Caco-2 cells were infected with STM-WT, STM-?sopF or STM-?sopF/psopF at 24 hpi. Western blot analysis of whole cell lysates with specific antibodies to (e) pro-Caspase-8, cleaved Caspase-8, PDK1 and pRSK; (f) GSDMD/GSDMD-NT, GSDME, GSDME-NT, pro-Caspase-3/cleaved Caspase-3; pMLKL and the control Actin. (g, h) Quantification of western blot analysis above. Data were compared by one-way ANOVA. Values are expressed as the means ± SD, and statistically significant differences are indicated. ***P < .001; **P < .01; *P < .05; ns: not significant. Data were from at least three experiments.
Fig 2: SopF regulates the PANoptosis of intestinal epithelial cells in host defense against S. Typhimurium infection. (a, b) Caco-2 cells (a) or NCM460 (b) cells were infected with STM-WT, STM-?sopF or STM-?sopF/psopF at an MOI of 100. LDH release were detected at 2 hpi, 8 hpi, 16 hpi and 24 hpi. (c-h) Streptomycin-pretreated C57BL/6 mice were orally infected with 5 × 107 CFU of STM-WT, STM-?sopF or STM-?sopF/psopF. IECs were isolated from ceca of mice at 12 hpi, 48 hpi and 120 hpi. Western blot analysis of isolated IEC lysates with specific antibodies to (c) GSDMD/GSDMD-NT, GSDME, GSDME-NT; (e) pro-Caspase-3/cleaved Caspase-3; (g) pMLKL and the control Actin. (d, f, h) Quantification of western blot analysis above. (i-h) Caco-2 cells were infected with STM-WT, STM-?sopF or STM-?sopF/psopF at an MOI of 100 at 24 hpi. Western blot analysis of whole cell lysates with specific antibodies to (i) GSDMD/GSDMD-NT, GSDME, GSDME-NT; (k) pro-Caspase-3/cleaved Caspase-3; (m) pMLKL and the control Actin. (j, l, n) Quantification of western blot analysis above. Data were compared by one-way ANOVA. Values are expressed as the means ± SD, and statistically significant differences are indicated. ***P < .001; **P < .01; *P < .05. Data were from at least three experiments.
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