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. 2020 Sep 4;295(48):16328–16341. doi: 10.1074/jbc.RA120.015110

Figure 6.

Figure 6.

Vgf is a Sox9 target gene. A, schematic representation of murine Vgf promoter, highlighting the putative Sox9 binding site. B and C, HEK293 cells were stably transfected with empty plasmid or Sox9 construct. The mock transfected cells (−) and Sox9 expressing cells (+) were then used for luciferase-based reporter assays. The Vgf promoter sequences (−2000bp from transcription start site (TSS)) was cloned in a luciferase reporter construct. Mock (−) and Sox9 (+) expressing cells were transiently co-transfected with reporter Renilla luciferase constructs (empty, Vgf, or Vgfmut) and reference Cypridina luciferase (normalizing control), followed by measurement of luciferase activity at 24 h. The normalized luciferase activity of mock (−) group was then compared with the Sox9 (+) group. The results show that the Sox9 can activate transcription from the Vgf promoter. In the Vgfmut construct, the Sox9 binding site was mutated from ATTGTT to AACAAT. D, Sox9 ChIPs were carried out from the renal tissues of control and Sox9PT−/− mice undergoing ischemia-, cisplatin nephrotoxicity-, and rhabdomyolysis-associated AKI. Subsequent qPCR analysis using primers specific for the murine Vgf promoter region showed that Sox9 can bind to Vgf promoter in vivo. E–G, qPCR-based gene expression analysis was carried out in the renal tissues from littermate control and Sox9PT−/− mice at indicated time points after induction of kidney injury. The injury-induced Vgf mRNA up-regulation was suppressed in the Sox9PT−/− mice. H, immunoblot analysis of renal cortical tissues showed that AKI-induced Vgf protein induction is suppressed in the Sox9PT−/− mice. Blots are representative of three independent experiments. In all the bar graphs (n = 8–9 biologically independent samples), experimental values are presented as mean ± S.D. The height of error bar = 1 S.D., and p < 0.05 was indicated as statistically significant. One-way ANOVA followed by Tukey's (C) or Dunnett's (D–G) multiple-comparison test was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001.