Extended Data Fig. 10: The role of mtROS production by RET in NLRP3 inflammasome activation.
BMDMs from wild-type (WT) and ND6P25L mice stimulated with LPS for 4 h or primed with LPS for 4 h followed by treatment with 5 mM ATP or 15 μM nigericin for 1 h. a,b, Western blot and quantification of NLRP3 and tubulin (n = 3). Blots show three biological replicates for each condition and data are mean normalized to NS and tubulin loading control +/- S.E.M. of biological replicates. c-f, Western blot and quantification of pro- and mature IL-1β and tubulin in cell lysates and protein released into cell supernatant (n = 3). Blots show three biological replicates for each condition and data are mean normalized to NS and tubulin loading control +/- S.E.M. of biological replicates. g-i, Western blot and quantification of caspase-1 p45 and vinculin in cell lysates and caspase-1 p20 released into cell supernatant (n = 3). Blots show three biological replicates for each condition and data are mean normalized to NS and vinculin loading control +/- S.E.M. of biological replicates. j, Western blot of ASC protein with or without treatment with cross-linking agent BS3 (n = 3). Blot shows three biological replicates for each condition. k-m, Western blot and quantification of intact and cleaved gasdermin D (GSDMD) (n = 3). Blots show three biological replicates for each condition and data are mean normalized to NS and vinculin loading control +/- S.E.M. of biological replicates. n, The ATP/ADP ratio measured per million cells in nonstimulated (NS) BMDMs and BMDMs stimulated with LPS for 4 h from WT and ND6P25L mice (n = 3). Data are mean +/- S.E.M. of biological replicates. P values displayed above graphs calculated using two-tailed Student’s t-test for paired comparisons or one-way or two-way ANOVA for multiple comparisons. o, Gating strategy for neutrophils as analysed by flow cytometry.
