Control nonspecific sirnas

BMDMs were transfected with small INTERFERing RNAs. Briefly, cells were plated in 48-well plates (at a density of 5 × 10⁵ cells per well) and then were transfected with 50 nM siRNA through the use of INTERFERin (Polyplus) according to the manufacturer’s guidelines. Control nonspecific siRNAs and the specific siRNAs were purchased from Sigma-Aldrich. The siRNAs used were: Drp1 a (5′GGAAUAAUUGGAGUAGUUAdTdT3′), Drp1 b (CUGUCAAUUUGCUAGAUGUdTdT), DDX33 a (GCAAGAAUAUGCUGCUAGUdTdT), DDX33 b (CCCAAAUGUGCUCACCUUUdTdT), RIPK1 a (CACAAUCCUUUCUUACACAdTdT), RIPK1 b (GGAAGAUAUUGUGAGCGGAdTdT), NLRP3 a (GAUCAACCUCUCUACCAGAdTdT), NLRP3 b (GUGUUGUCAGGAUCUCGCAdTdT), GSDMD a (GAUUGAUGAGGAGGAAUUAdTdT), GSDMD b (CUGCUUAUUGGCUCUAAAUdTdT).

HEK293T cells were transfected by the calcium phosphate precipitation method, whereas HeLa cells and MEFs were transfected with the Lipofectamine RNAiMAX (Invitrogen, Life Technologies), according to the manufacturer’s instructions. siRNAs were used at a final concentration of 20 nM. Control non-specific siRNAs and the specific siRNAs were purchased from Sigma-Aldrich. The siRNAs used were: OPTN 1 (5′ GCUUCAAGAGGCACACACAdTdT 3′), OPTN 2 (GUUUGAGAUGCAAAGCAAAdTdT), mNEMO (GCGAGUUCAACAAGCUGAAdTdT), OPTN A (GAGUCAUGAGAAUGAGAAAdTdT), OPTN B (CCAUGAAAGGGAGAUUUGAdTdT), OPTN C (CCAAAGAAAGAGUUUCAGAdTdT), OPTN D (GAAAGCAUGCUAUCAGAAAdTdT), OPTN E (CCAAGAAUUACUUCGAACAdTdT), and hNEMO (GAGUCGUUGGAGGCUGCCACUAAGdTdT).