Extended Data Fig. 10: SERPINB6B controls antigen presentation and blocks CTSG and Caspase-1 activity.
From: LncRNA Malat1 suppresses pyroptosis and T cell-mediated killing of incipient metastatic cells
a, Flow cytometry data of B2m, H-2Kd and H-2Dd at indicated condition (n = 3 independent experiments). b, Percentage of LDH release over control value in control, Malat1 KO, and Malat1 KO 4T1 cells overexpressing S6b exposed to Nigericin and ATP. LDH release was measured by using the CytoTox 96 assay (n = 3 independent experiments). c, RT-qPCR analysis of Gsdma, Gsdmc, Gsdmd, and Gsdme in 4T1-TGL cells (n = 3 independent experiments). d, Immunoblotting of GSDMD and GSDME in WT and Malat1 KO 4T1 cells. e, Percentage of LDH release over control value in WT, Malat1 KO, and Malat1 KO 4T1 cells overexpressing S6b exposed to raptinal for 4 hours (n = 5 independent experiments). f, HEK-293 cells were transfected with mouse Flag-Gsdmd and lysed without protease inhibitors. 10 μg of total proteins was incubated with the indicated concentrations of the indicated proteases for 1 h at 37 °C in 25 μl of reaction buffer. Samples were resolved on SDS-PAGE and subjected to immunoblotting with anti-Flag antibodies. g, HEK-293 cells were transfected with mouse Flag-Gsdmd in combination with HA-S6b or HA-S3g and lysed without protease inhibitors. Total proteins (10 μg) were incubated with increasing concentrations of proteases (25, 50, 100, 200 ng of CTSG; 0.002, 0.02, 0.2, 2 U of Casp-1; 0.05, 0.5, 5, 50 U of Casp-11) for 1 h at 37 °C, resolved on SDS PAGE, and subjected to immunoblotting with anti-Flag antibodies. h, RT-qPCR analysis of CtsG expression in 4T1-TGL cells overexpressing CtsG (n = 3 independent experiments). i, RT-qPCR analysis of CtsG expression in Malat1 KO 4T1 cells overexpressing Serpinb6b or not (n = 3 independent experiments). j, Malat1 KO 4T1 cells were transduced with mirE-shRNAs targeting Gsdmd or control shRNA and subjected to immunoblotting as indicated. P values in a,b,f, and i were calculated using a one-way ANOVA with multiple comparisons, and in c and h using a two-tailed, unpaired Student’s t-test. Data are presented as mean values +s.e.m (a, b, f, i) or +s.d. (c, h).
