Figure 4. mTORC1 acts downstream of GSDMD cleavage to promote pore formation and pyroptosis.

(A–C) HEK293 cells were co-transfected with plasmids encoding hNT-GSDMD (I104N) and TSC-1. Normalized PI fluorescence (A), extracellular LDH activity (B) and protein abundance (C) were measured 20 hours post-transfection.

(D) MyD88 L265P-driven NF-κB dual-luciferase system response in HEK293 cells transfected with TSC-1 24 hours after transfection. Response of MyD88 L265P transfected cells over empty vector transfected cells is shown.

(E, F) PI uptake (E) and LDH release assessment (F) of LysM-CRE WT, Rictor-deficient, and Raptor-deficient iBMDMs, primed with recombinant IFNβ for 3 hours then electroporated with PBS or LPS.

(G) Frequency of Annexin-V and 7-AAD positive cells by flow cytometry from LysM-CRE WT, Rictor-deficient, and Raptor-deficient iBMDMs after 8 hours of 1 μM Staurosporine (STS) treatment.

(H, I) PI uptake (H) and LDH release (I) from LysM-CRE WT, Rictor-deficient, Raptor-deficient, C57/BL6J WT, NLRP3-deficient, and GSDMD-deficient iBMDMs, unstimulated or infected with S. Typhimurium at an MOI of 10 for 1 hour.

(J) Western blot of the proteins indicated in LysM-CRE WT, Rictor-deficient, and Raptor-deficient iBMDMs, after these cells were primed with IFNβ and then electroporated with PBS or LPS.

(K) Western blot of the proteins indicated in LysM-CRE WT, Rictor-deficient, Raptor-deficient, C57/BL6J WT, NLRP3-deficient, and GSDMD-deficient iBMDMs, after these cells were unstimulated or infected with S. Typhimurium at an MOI of 10 for 1 hour.

All quantifications represent mean and SEM of 3 independent experiments. Two-way ANOVA was used for analysis. See also Figure S4.