Figure 4.

Effect of vitamin D3 on protein expression of DDIT4, TSC1, and TSC2 in cultured human uterine fibroid cells. A, HuLM cells were serum starved and treated with increasing concentrations of vitamin D3 (0, 1, 10, 100, and 1000 nM) for 48 hours, as described above. Equal amounts of each cell lysate were analyzed by Western blots using anti-DDIT4, anti-TSC1, and anti-TSC2 antibodies. β-Actin Western blot was used as loading control. B and C, Immunofluorescence analyses were performed using both HuLM cells (B) and human PUF cells (C) cultured on glass coverslips and treated with increasing concentrations of vitamin D3 (0, 10, 100, and 1000 nM) for 48 hours. Cells were fixed, permeabilized, and stained with anti-DDIT4 antibody (1:50 dilution) followed by incubating with carbocyanine 3-conjugated antirabbit secondary antibody. DDIT4 staining (red) was monitored by fluorescence microscopy. Nuclei of cells were stained with 4′,6-diamino-2-phenylindole. Pictures were taken at ×200 magnification.