Fig. 2

ERRα inhibits caspase-1/GSDMD signaling via transcriptional binding with NLRP3. A Interaction between HIF-1α and ERRα was found in the STRING protein interaction database. B Interaction between HIF-1α and ERRα was verified using the co-immunoprecipitation (Co-IP) analysis of HEC-1A^−ovERRα cell lysates. The corresponding antibodies (out-put IP: anti-HIF-1α-antibody and anti-ERRα-antibody) could precipitate both ERRα and HIF-1α, respectively, in the HEC-1A^−ovERRα cell lysates. C Effects of ERRα regulation on the levels of pyroptosis-related proteins (NLRP3, GSDMD-N, caspase-1, IL-1β, and IL-18) in EC cells was analyzed using WB; β-actin was used as the loading control. Untreated KLE and HEC-1A cells and the empty vector-transfected KLE and HEC-1A cells treated with DDP were used as the control groups. D GO analysis showing the enriched biological process in EC cells overexpressing ERRα, HEC-1A cells were used as the control group. E KEGG pathway analysis for the genes that showed ERRα-binding peaks in their promoter regions. F–H Dual-luciferase reporter assay to assess the interaction between ERRα and NLRP3. (F) 293 T cells were co-transfected with ERRα transcription factor, NLRP3 promoter labeled with luciferase reporter, and the Renilla luciferase control. The negative-control (NC) group was NLRP3-NC with ERRα-NC and NLRP3 with ERRα-NC. (G) Three NLRP3 promoter fragments ligated to the pGL3-basic plasmid, named NLRP3-1 (-400 to + 21), NLRP3-2 (-800 to + 21), and NLRP3-3 (-1200 to + 21), were co-transfected with ERRα transcription factor; the three NLRP3 promoter fragments with ERRα-NC were used as the control. (H) NLRP3-2-MUT and wild-type NLRP3-2 and their empty vectors were co-transfected with ERRα transcription factor. The vector-control group was NLRP3-2 with ERRα-NC and MUT with ERRα-NC. The luciferase activity was measured and normalized to that of the Renilla luciferase control 48 h post-transfection and the relative luciferase activity was determined. I Schematic diagram of putative ERRα-binding sites, as predicted by the online program JASPAR, located in the NLRP3 promoter region (P1–P5). The sequence TCAAGGTCA of ERRα is placed on the left. J CUT&Tag and sequencing analysis of the distribution map of ERRα peaks in gene functional regions. K NLRP3 promoter occupancy in HEC-1A cells was evaluated. ERRα was immunoprecipitated using an anti-Flag antibody. Data are representative of three independent experiments and shown as the mean ± SD. *p < 0.05; **p < 0. 01; ***p < 0.001; ****p < 0.0001. Abbreviations: ERRα, estrogen-related receptor alpha; HIF-1α, hypoxia-inducible factor-1 alpha. Co-IP, co-immunoprecipitation. CON; control empty vector. NC: negative control. MUT: mutation