Figure 2.

(A,B) Effect of TGF-β on PTHrP promoter activity in AR42J and irPSCc3 cells. Cells were transfected with the indicated promoter-driven luciferase reporter constructs plus Renilla luciferase construct, then treated with TGF-β (3 ng/ml). Luciferase activity was measured after 2 h. Empty vector control values were subtracted from the respective firefly and Renilla luciferase values. Values were then normalized to Renilla luciferase activity, and are expressed as the firefly/Renilla ratio. P = promoter. Each bar is the mean ± SEM of three experiments. ***, P < 0.001 vs. −TGF-β. (C) ChIP assay of chromatin from AR42J and irPSCc3 cells treated with TGF-β. Cells were treated with TGF-β for 1 h. Immunoprecipitation was performed using an anti-Smad2/3 antibody (Smad) and an anti-acetyl histone H3 antibody (N-AcHis). Normal IgG was used as antibody control. DNA enriched by ChIP was subjected to real-time PCR using a PTHrP P3 promoter primer. Data are presented as fold-enrichment vs. the IgG value, set arbitrarily as 1.0. Each bar is the mean ± SEM of three experiments. ***, P < 0.001 vs. IgG control, #, P < 0.001 vs. vehicle control. (D,E) Effect of inhibiting Smad3 signaling in AR42J and irPSCc3 cells on TGF-β-mediated upregulation of PTHrP mRNA levels. Cells pre-treated with SIS3 (10 μM) for 1 h were then treated with TGF-β for 2 h. PTHrP mRNA levels were measured by reverse transcription/real-time PCR. Values are expressed relative to the vehicle control value, set arbitrarily at 1.0. Each bar is the mean ± SEM of three independent experiments. ***, P < 0.001 vs. control; #, P < 0.001 vs. TGF-β alone.