Fig. 4

Docetaxel is crucial for binding of Smad3 to the HIF-1α promoter and subsequent HIF-1α transactivation. A PC-3M cells were grown to 70–80% confluence, serum starved for 24 h, then stimulated with or without 5 uM docetaxel for 1 h, whole cell lysates were immunoprecipitated (IP) with antibodies targeting endogenous Smad3 and co-precipitates with HIF-1α were detected by immunoblotting. B PC-3M IE8 (left panel) and PC-3M (right panel) cells were transfected with plasmid, including pCDNA3.1 and pCDNA3.1-Smad3, combined with a vector containing the HIF-1α promoter sequence driving the firefly luciferase reporter (HIF-1α -luc) in conjunction with a control Renilla luciferase expression vector. At 24 h after transfection, cells were serum starved for an additional 12 h, followed by 5 uM docetaxel treatment for 16 h. Luciferase reporter activity is presented as the fold activation relative to Renilla luciferase activity. Data represented the mean ± s.d. of three independent experiments and analyzed by two-way ANOVA with multiple comparisons, followed by Bonferroni post hoc test for significance. **P < 0.01, ***P < 0.001. C PC-3M cells were serum starved, and treated as indicated for 1 h, the whole cell lysates were immunoprecipitated with an anti-Smad3 antibody, co-precipitating chromosome fragments binding to Smad3 in vivo were amplified and quantified by real-time PCR. Results are presented as a ratio of the immunoprecipitated product to the input product. Left panel: real-time PCR of the Smad3-enriched HIF-1α promoter region. Right panel: real-time PCR of a nonspecific region corresponding to the exon of the HIF-1α gene enriched by Smad3 (negative control). Data represented the mean ± s.d. of three independent experiments and analyzed by two-way ANOVA with multiple comparisons, followed by Bonferroni post hoc test for significance.