Figure 1.
VDR expression and subcellular distribution in HaCaT cells.
Cell were incubated for 24 hours alone (control, C) or with 1 or 100 nM 1,25D3 (D1 or D100) and harvested. (A) After subcellular fractionation procedures 50 µg of proteins from total extracts (TOT) and soluble fraction (SOL), and 10 µg of proteins from mitochondria (MIT) and nuclear extracts (N) were separated by SDS-PAGE and analysed by western blotting for VDR expression. Equal loading and quality of samples was confirmed by reprobing the membranes with antibodies anti actin, VDAC (mitochondrial marker) and PARP (nuclear marker). (B) Same amount of total extracts and mitochondrial fractions were analysed by western blotting for RXRα expression. (C) 10 µg of proteins from mitochondria (MIT) and nuclear extracts (N) of untreated cells (ctrl) or cells infected with shRNA control and shRNA anti-VDR were analysed by western blotting for VDR expression and afterwards for loading uniformity.
Figure 2.
VDR association with RXRα and PTP proteins.
(A) Mitochondrial extracts from untreated HaCaT cells were immunoprecipitated with anti-VDR and anti-RXRα rabbit antibody and detection by western blotting was performed with anti-VDR or anti-RXRα biotinylated antibodies and with anti-VDAC rabbit antibody. (B) The interaction between VDR, RXRα and StAR was investigated in mitochondrial fraction by immunoprecipitation with anti-StAR rabbit antibody followed by western blotting and detection with anti-VDR and anti-RXRα biotinylated antibodies. In every assay a 10% input was used as a positive control and immunoprecipitation with normal IgG as negative control.
Figure 3.
Analysis of mitochondrial translocation of VDR in presence of cyclosporin A.
HaCaT cells were treated with cyclosporin A (CsA), cycloheximide (CHX) or 100 nM 1,25D3 (Vit.D) as indicated and 30 µg of mitochondrial proteins were analysed by western blotting for VDR, RXRα and p53 expression. Bands were quantified, normalized for loading as a ratio to VDAC expression and data plotted on graph as percentage of control. Data represent the mean ± S.D of three independent experiments. *p<0.05 and **p<0.001 compared to control. # p<0.05 vs CHX CsA 18 h+vit.D 24 h.
Figure 4.
Effect of squalestatin treatment on StAR and VDR expression.
HaCaT cells were treated with squalestatin (SQ) and 100 nM 1,25D3 (D) or left untreated (control, C), harvested and 50 µg of total lysates (tot) or 30 µg of mitochondrial proteins (mitoc) were analysed by western blotting for StAR and VDR expression. Bands were quantified, normalized for loading as a ratio to actin expression (tot) or VDAC expression (mitoc) and data plotted on graph as percentage of control. Data represent the mean ± S.D of three independent experiments. (A) Analysis of StAR expression in mitochondrial fractions after 48 or 72 hours of treatment with SQ, 24 hours with vit. D or cotreatment in which vit. D was added in the last 24 hours (SQ+D). (B) VDR expression after squalestatin treatment in a time course experiment. (C) VDR expression after 72 hours of the same treatments as in (A). §p<0.01 and **p<0.001 compared to control.
Figure 5.
Western blot analysis of the expression of VDR and StAR upon dexamethasone treatment.
30 µg of mitochondrial proteins (A) or whole lysates (B) from untreated HaCaT cells (ctrl) and cells treated for 72 h with dexamethasone (Dex) were analysed by western blotting using an antibody anti-VDR, followed by immunostaining with anti-StAR and finally with anti-VDAC or anti-actin antibody for loading control. The blots are representative of a set of three independent experiments.
Figure 6.
Effect of genetic silencing of StAR on VDR expression.
Subconfluent HaCaT cells were infected with lentiviral StAR shRNA particles to silence the endogenous StAR expression. Mitochondrial fractions from untreated HaCaT (ctrl) and cells infected with shRNA control and StAR were analysed by western blotting for StAR and VDR expression. VDR levels were also evaluated in total lysates. VDAC was used as internal control for protein loading.