Fig. 4.
Sufu mediates SMC gene expression by modulating GLI1 nuclear translocation. a, b Sufu knockdown upregulated SMC gene expression. AdSPCs were infected with non-target (sh-NT) or Sufu (sh-Sufu) shRNA lentivirus, followed by iSMC differentiation. Total RNAs and proteins were harvested and subjected to RT-PCR (a) and Western blot (b) analyses, respectively. c SMC gene promoter activity was upregulated by Sufu inhibition. AdSPCs infected with sh-NT or sh-Sufu lentivirus were transfected with SMC gene promoters (pGL3-SMαA or SM22α). Two days later, total cell lysates were harvested and subjected to luciferase activity assays. d, e Sufu knockdown increases GLI1 nuclear translocation. AdSPCs were subjected to similar treatments described in a and b. Total cell lysate and nuclear proteins were extracted and subjected to Western blot analysis. f Two GLI1 binding sites within the SMαA gene promoter region are required for SMαA gene upregulation by Sufu knockdown in AdSPCs. The potential wild-type binding site (WT) of GLI1 within the SMαA gene promoter region and its mutants (GLI1mut) is depicted in this illustration (top). AdSPCs infected with sh-NT or sh-Sufu lentivirus were transfected with wild-type and mutated SMαA gene promoter reporters as indicated. Luciferase activity assay was measured at 48 h post-transfection. g Sufu inhibition increases GLI1 enrichment within the SMαA gene promoter. ChIP assays were performed using antibodies against GLI1 or normal IgG. PCR amplifications of the adjacent regions were included as an additional control for specific promoter DNA enrichment. The data presented here are representative (left panel in b, and d) or mean ± S.E.M. (a, c, e–g and right panel in b) of five independent experiments (n = 5). *P < 0.05, **P < 0.01 (vs sh-NT, unpaired t test)