FIG. 7.

Ablation of FoxO expression attenuates or blocks inhibition of SCC25 cell proliferation by 1,25D. (A) siRNA-mediated knockdown of FoxO3a or FoxO4. SCC25 cells were transfected with siRNAs recognizing FOXO3A, FOXO4, or control siRNA, and expression of FoxO proteins was assessed by Western blotting. (B) Ablation of FoxO expression does not affect regulation by 1,25D of a VDRE-containing promoter. SCC25 cells were transfected with the VDRE3/tk-luc expression vector along with control siRNAs or siRNAs directed against FOXO3A and/or FOXO4, as indicated. Cells were treated with vehicle or 1,25D, and extracts were assayed for luciferase activity. (C) Effects of ablation of FoxO expression on regulation by 1,25D of target gene CYP24. SCC25 cells were transfected with control or FoxO-specific siRNAs, and transfected cells were treated with 1,25D as indicated. Expression of CYP24 was analyzed by qRT-PCR. (D) Effects of ablation of FoxO expression on regulation by 1,25D of FoxO target genes. SCC25 cells were transfected with control or FoxO-specific siRNA, and transfected cells were treated with 1,25D as indicated. Expression of FoxO target genes was analyzed by qRT-PCR. (E) Effects of FoxO knockdown on regulation by 1,25D of cyclin D2 expression. SCC25 cells were transfected with control or FoxO-specific siRNAs, and transfected cells were treated with 1,25D for 8 h. Cyclin D2 expression and actin control were assessed by Western blotting. Error bars represent SD. *, P < 0.05.