Fig. 6: GSDMD deficiency inhibits the activation of the stat5a/CXCL1/neutrophil pathway. | Nature Communications

Fig. 6: GSDMD deficiency inhibits the activation of the stat5a/CXCL1/neutrophil pathway.

From: Gasdermin D aggravates a mouse model of radiation-induced liver disease by promoting chemokine secretion and neutrophil recruitment

Fig. 6

A Identification of promoters and enhancers of CXCL1 with integrative analysis of transcriptomic sequencing and database within the GeneCards Suite (http://wwww.genecards.org/). B Heatmap of expression of the indicated genes from (A) (n = 5/group). C, D Primary hepatocytes were pretreated with or without 25 μM STAT5A inhibitor IST5-002 for 6 h prior to 8 Gy irradiation. After 24 h, the translocation inhibited ability were determined by the WB assay (C); the culture medium level of CXCL1 was assayed by ELISA (D). Vehicle vs IST5: Ctrl, p = 0.008; IR, p = 4.98E-06. E Luciferase activity assay was performed to measure the binding of STAT5A at the GsdmdFL/FL or the promoter region with mutations (Mut) of Cxcl1 gene with or without IR in HEK 293 T cell. WT-Ctrl vs WT-IR: p = 6.66E-13. F Quantification of neutrophil migration in response to irradiated hepatocytes pretreated with IST5-002 or not. Vehicle vs IST5: Ctrl, p = 0.003; IR, p = 1.71E-05. G Immunoblot analysis of STAT5A and P-STAT5A from GsdmdFL/FL and Gsdmd∆Hep primary hepatocyte exposed to 8 Gy irradiation. H Protein level of STAT5A and P-STAT5A in cytoplasmic extracts and nuclear extracts from 8 Gy irradiated GsdmdFL/FL and Gsdmd∆Hep primary hepatocytes were determined by Immunoblot. I Co-IP analysis for physical interaction of GSDMD and STAT5A in hepatocytes. J ChIP assays were performed to detect the promoter sites of STAT5A binding to Cxcl1. IgG as a negative control for ChIP assay. GsdmdFL/FL: IgG vs stat5a, p = 1.12E-08; stat5a: GsdmdFL/FL vs Gsdmd∆Hep, p = 1.75E-08 C-J, n = 3 biologically independent mice per group. K, L Immunoblot analysis of STAT5A and P-STAT5A from GsdmdFL/FL and Gsdmd∆Hep mice (n = 1, no IR; n = 5, IR). M Immunoblot analysis of STAT5A and P-STAT5A of IR-Gsdmd∆Hep mice administered with AAV9-Control and AAV9-GSDMD-FL (n = 4/group). p values were using two-way ANOVA with Fisher’s LSD multiple comparisions test (D-F and J). No data were excluded from the analyses. Data are expressed as mean ± SEM. Source data are provided as a Source Data file.

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