Fig. 3.

(A,B) Anti-proliferative effects of 1,25D occur in the presence of a VDR. Cell density values, as measured with the CellTiter96 AQueous non-radioactive cell proliferation assay (Promega), were obtained every 24 h over a period of 5 days from SaOS-2 cell cultures treated either with 10 nM 1,25D for 15 min on day 0 (A, transient), or grown in continuous presence of hormone (B, sustained). Squares represent data obtained from control Non-T cells in the absence of 1,25D. Inverse triangles, circles, and upright triangles represent data obtained from siRNA VDR knock-down, control Non-T (3) and control V-T (4) cells in the presence of 10 nM 1,25D; n=5, *, p<0.001. (C,D) Transient and sustained 1,25D treatment induces JNK activation and c-Fos upregulation only in cells expressing a VDR. Western blots obtained for activated (p-) JNK, p-c-Jun, and c-Fos in native Non-T, control V-T, and VDR knock-down (siRNA-T) SaOS-2 cells treated with 10 nM 1,25D for 15 min (transient, C) or 3 days (sustained, D). Activated JNK and c-Jun proteins were detected with commercial antibodies raised against phosphorylated residues. VDR protein expression levels are also shown for the same cell lysates.