Control of TCF-4 Expression by VDR and Vitamin D in the Mouse Mammary Gland and Colorectal Cancer Cell Lines (original) (raw)
Figure 2
TCF-4 is increased by 1,25(OH)2D3 in a VDR-dependent manner in human colorectal cancer cells.
(A) CYP24-luc activity in VDRK240−/− (VDR −/−) and VDR145+/+ (VDR +/+) cells that were transfected with GFP or VDR and treated with 10−7 M 1,25(OH)2D3 or EtOH-control for 24 hours as indicated. RLU = Relative Light Units. (B) Reverse Transcriptase PCR analysis of mouse CYP24A1 (top panel), mouse and human VDR (middle panels) and β-actin (bottom panel) transcripts in response to exogenous human VDR expression and 1,25(OH)2D3 treatment as described in part A. (C) Various colorectal cancer cell lines were incubated for 24 hours in 10−7 M 1,25(OH)2D3 or EtOH, as indicated. Cellular proteins were assayed for TCF-4 expression (upper panel) and GAPDH was monitored for equal lane loading (lower panel). Both TCF-4 bands represent different isoforms of TCF-4 that have different length C-termini. (D) CaCo2 cells were transfected with different amounts of VDR or siRNA for 24 hours and treated with 10−7 M 1,25(OH)2D3 or EtOH for a subsequent 24 hours, as indicated, and probed for TCF-4. NT: Non-targeting. (E) Densitometric analysis of three western blots as indicated in part A. Data were plotted relative to each EtOH-treated control. p-values were generated by one-tailed t-test: * p<.05; ** p<.005; *** p<.0005 (F) CaCo2 cells were transfected for 24 hours with VDRE-luc, Renilla and siRNA and treated for a subsequent 24 hours with 10−7 M 1,25(OH)2D3 as indicated. RLU: Relative Light Units. (G) CaCo2 cells were transfected with siRNA for 24 hours and treated for a subsequent 24 hours with 10−7 M 1,25(OH)2D3 or EtOH as indicated. CYP24A1 transcripts were assayed by qPCR. Data are normalized to GAPDH expression and plotted relative to each EtOH control.