Fig. 4

Cortisol enhances the interaction between GR with the CXCL1 promoter. A CXCL1 mRNA expression levels in M2-polarized RAW264.7 cells were measured by qPCR under cortisol treatment with various times and doses. B M2-polarized RAW264.7 cells were transfected with CXCL1 promoter-luciferase reporter plasmids that carried Gaussia luciferase (GLuc) and secreted alkaline phosphatase (SEAP) reporter gene. Gluc and SEAP activity of M2-polarized RAW264.7 cells treated with cortisol, GR antagonist (RU486) or shGR were measured using the luminometer. Results were displayed as ratios of GLuc and SEAP luminescence intensities. C Schematic diagram of the potential GR binding site on CXCL1 promoter predicted by hTFtarget database. The interaction between the CXCL1 promoter and GR was detected by Chromatin immunoprecipitation (ChIP) assay under cortisol, GR antagonist (RU486) or shGR treatment. D Schematic illustration of CXCL1 promoter mutation site (upper panel). Mutated CXCL1 promoter-luciferase reporter plasmids were introduced into the M2-polarized RAW264.7 cells. The luciferase activity was compared with wild-type CXCL1 promoter (lower panel). Data are represented as the Mean ± SD. For statistical analysis, unpaired t-tests (B, C, D), and one-way ANOVA with Dunnett post hoc test (A, B, C), were applied. ^*P < 0.05, ^#P < 0.01