Fig. 5: GSDMD regulates hepatocytes release CXCL1 to recruit neutrophil.

A Immunofluorescence visualization of CXCL1 (green), HNF4α (red), ɑ-SMA (red) in liver sections from Ctrl and IR-treated mice (n = 5/group). (Scale bars, 200×, 100 μm). B Double-immunostaining for CXCL1 and GSDMD-FL in primary hepatocytes 24 h after irradiation. Confocal analysis demonstrates coexpression of CXCL1 and GSDMD-FL in hepatocytes receiving radiation. Scale bars, 200×, 50 μm (n = 5/group). C Cell viability was assessed by Annexin-V⁺/PI⁺. D ELISA assay of CXCL1 release in culture supernatants from Gsdmd^FL/FL and Gsdmd^∆Hep hepatocyte accepted irradiation or not. IR: Gsdmd^FL/FL vs Gsdmd^∆Hep, p = 0.0004. C, D each sample was from 3 biologically independent replicates. E Immunoblot analysis of caspase-1, cleaved-caspase-1, GSDMD-FL/N and CXCL1 expression from Gsdmd^FL/FL primary mouse hepatocytes treated or untreated with 20 μM Ac-YVAD-cmk, analyzed at 12 h following 8 Gy irradiation (3 biologically independent replicates). F ELISA assay of CXCL1 release in culture supernatants from hepatocytes pretreated without or with 20 μM Ac-YVAD-cmk. IR: Vehicle vs Ac-YVAD-cmk, p = 1.42E-07. G, H Quantification of neutrophil migration. Quantification of neutrophil migration in response to irradiated Gsdmd^FL/FL hepatocytes pretreated with anti-CXCL1 antibody or not (G), p = 0.003; Quantification of neutrophil migration in response to irradiated Gsdmd^∆Hep hepatocytes pretreated with CXCL1 RP or not (H), p = 0.002. F–H, n = 3 biologically independent mice per group. P values were using two-way ANOVA with Fisher’s LSD multiple comparisions test (D, F) and unpaired two-sided Student’s t test (C and G, H). No data were excluded from the analyses. Data are expressed as mean ± SEM. Source data are provided as a Source Data file.