Fig. 3

Smad6 promotes STAT3 activation through inhibition of PIAS3. a, b Nuclear-Smad6 regulated STAT3 downstream genes expression in GBM cells. The expression of proteins downstream of STAT3 signaling (c-jun, CCND1, and Sox2) were analyzed by western blot in glioma cells with Smad6 KD (a) or nuclear-Smad6 OE (b). The quantification of indicated proteins was listed. c Nuclear-Smad6 enhanced the enrichment of STAT3 on the promoter of STAT3 downstream targets. The enrichment of STAT3 binding to the promoter of target genes (SOX2 and CCND1) was examined by ChIP-qPCR in glioma cells with nuclear-Smad6 OE (left panel) or KD (right panel) (n = 3). d Smad6 KD or e nuclear-Smad6 OE did not influence the phosphorylation (Y705) of STAT3, but regulated PIAS3 expression. f Nuclear-Smad6 rescued STAT3 transcriptional activity that was inhibited by PIAS3 (n = 3). Luciferase assay of STAT3 transcriptional activity in HEK293 cells transfected with HA-PIAS3 and/or indicated doses (μg) of Flag-Smad6-NLS. IL-6 (25 ng ml⁻¹) was added 6 h before the assay. Data were represented as means ± SD and analyzed using two-tailed Student’s t-test. **P < 0.01