Fig. 4.

PRL effect on 1α,25(OH)₂D₃-induced transcription of both VDR (A) and osteocalcin (B) genes. Ros 17/2.8 osteosarcoma cells were cultured in DMEM with 10% FBS and 1% PS and then incubated in the absence of FBS for 24 h before treatment with 1 nm 1α,25(OH)₂D₃ or this plus 200 ng/ml U-PRL or S179D PRL or diluent [control (CONT)] for 7.5 h. mRNA was quantified by real-time RT-PCR to produce panel A. ***, P < 0.001; ****, P < 0.0001 compared with 1,25D-treated cells. For panel B, cells were treated as above until the end of the incubation in the absence of FBS. They were then transfected with the hOCP/Renilla luciferase plasmid (hOCP/pMLuc-1) and/or control plasmids in the absence of FBS and PS for 12 h, followed by the treatment with daily refreshed 1 nm 1α,25(OH)₂D₃ or this plus 200 ng/ml U-PRL or S179D PRL or diluent (CONT) for 48 h. A Dual-Luciferase Reporter Assay System was used for the luciferase assay. ***, P < 0.001 compared with CONT; ♦♦♦, P < 0.001 compared with 1,25D-treated. Abbreviations as for previous figures.