Fig 1: E3 ubiquitin ligase MIB2 enhances GSDMD degradation via mediating the ubiquitination of GSDMD at K168. (A and B) HEK293T cells were transfected with HA-GSDMD and increasing amounts of Flag-MIB2. Cell lysates were collected for immunoblot analysis (A). Quantification of the relative protein level of GSDMD was shown in (B). (C to F) THP-1-derived macrophages were transfected with control siRNA (siCtrl) or MIB2 siRNA (siMIB2) for 48 h, pre-treated with LPS (200 ng/ml) for 3 h, then treated with DMSO (vehicle), MG132 (10 μM) or carfilzomib (100 nM), 3-methyladenine (3-MA, 10 mM), bafilomycin A1 (Baf A1, 0.2 μM), or chloroquine (CQ, 50 μM) for 6 h. Cell lysates were collected for immunoblot analysis (C and E). Quantitative analysis of the relative protein level of GSDMD was shown in (D) and (F), respectively. (G and H) THP-1-derived macrophages were transfected with siCtrl or siMIB2 (#1 and #2) for 48 h, then primed with LPS (200 ng/ml) for 3 h and followed by ATP (5 mM, 3 h) treatment. Cell lysates were collected for immunoprecipitation and immunoblot analysis (G). Quantitative analysis of the relative immunoprecipitated level of ubiquitin (Ub) was shown in (H). (I and J) HEK293T cells were transfected with HA-Ub and wild type (WT) Flag-GSDMD or Flag-GSDMD K168R, together with Myc-empty vector (EV) or Myc-MIB2 for 18 h, then treated with Baf A1 (0.2 μM) for 6 h. Cell lysates were collected for immunoprecipitation and immunoblot analysis (I). Quantitative analysis of the relative immunoprecipitated level of HA-Ub was shown in (J). (K and L) Immunoblot analysis of HEK293T cells transfected with Flag-GSDMD WT or Flag-GSDMD K168R mutant, together with HA-EV or HA-MIB2 (K). Quantitative analysis of the relative protein level of GSDMD was shown in (L). (M) In vitro binding assay of 6×His-MIB2 and Flag-GSDMD proteins purified from HEK293T cells, respectively. (N) In vitro ubiquitination assay was performed in the presence of Ub, E1, E2 (UbcH5a), Flag-GSDMD, and 6×His-MIB2. (O to Q) THP-1-derived macrophages were transfected with siCtrl or siMIB2 (#1 and #2) for 48 h, then primed with LPS (200 ng/ml) for 3 h and followed by ATP (5 mM, 3 h) treatment. Cell lysates were collected for immunoblot analysis (O). Cell death (P) and production of IL-1β (Q) were assessed by LDH (lactate dehydrogenase) release assay and ELISA analysis in the supernatants, respectively. In (A), (C), (E), (G), (I), (K) and (M) to (O), data are representative of 3 independent experiments with similar results. In (B), (D), (F), (H), (J), and (L), quantification of the indicated protein levels was determined by Image Lab software, data are presented as mean values ± SD, and P values were determined by unpaired 2-tailed Student’s t test of n = 3 independent biological experiments. In (P) and (Q), data are presented as mean values ± SEM and P values were determined by unpaired 2-tailed Student’s t test of n = 3 independent biological experiments.