Fig. 4

Characterization of IGFBP-3 expression induced by VD3 and DHT treatments. a Effect of dose-dependent VD3 treatment with or without DHT on IGFBP-3 induction. LNCaP cells were treated with VD3 alone at the indicated concentration or with VD3 and 1 nM DHT: (top) western blotting image of IGFBP-3 and (bottom) quantified graph of IGFBP-3 induction. Open circles; VD3 alone, filled circles; DHT + VD3. b Effect of temporal treatment of DHT/VD3 on IGFBP-3 induction: (top) schematic time course of temporal treatment of VD3 and (bottom) western blotting image of IGFBP-3. The ratio indicates the density of IGFBP-3 band normalized by corresponding βActin band. c Effect of mRNA transcription and protein synthesis on the stability of IGFBP-3 expression induced by DHT/VD3. LNCaP cells were treated with VD3/DHT for 1 day, followed by actinomycin D (ActD; 5 μM) or cycloheximide (CHX; 10 μM) for another day after VD3/DHT was washed out or left as is: (top) quantification graph of Igfbp-3 mRNA induction, (middle) western blotting image of IGFBP-3 and the combination of treatments (bottom). The ratio indicates the density of IGFBP-3 band normalized by corresponding βActin band. The all experiments were performed in serum-containing medium condition. The uncropped full-length blot images are presented in Supplementary Fig. 5C