1,25(OH)2D3 increases SIRT1 levels in CRC cells and VDR is required to ensure basal levels.
(A)-(D) HCT 116 or HT-29 CRC cells cultured under standard conditions and where indicated, treated with 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), 100 nM, added 24 hours before harvesting. (E)-(H) ShVDR cells were derived from HCT 116 by stable and specific knock-down of vitamin D receptor (VDR) using specific shRNA and are compared to ShControl cells that contain normal VDR levels (Larriba et al., 2011). Cell extracts were fractionated in all cases.
(A) Confocal imaging of SIRT1 (green) in CRC (HCT 116 and HT-29) cells and fluorescence intensity quantification of 3 independent experiments using ImageJ software. For each experiment 3 different fields were evaluated per slide. Scale bars:25µm.
(B) Western-blot analysis of 1,25(OH)2D3 effects on levels of SIRT1 in nuclear extracts (NE) from indicated CRC cells. Representative blots and statistical analysis using TBP as loading controls.
(C)-(E) RT-qPCR analysis of the effect of 1,25 (OH)2D3 in indicated colon cancer cells. Values normalized with endogenous control (18S RNA) are referred as fold induction over cells without 1,25(OH)2D3.
(C) Effect of 1,25 (OH)2D3 on SIRT1 gene expression.
(D) validation of 1,25 (OH)2D3 activity on the canonical target gene cytochrome P450 family 24 subfamily A member 1, CYP24A1, in HT-29 colon cancer cells.
(E) Requirement of vitamin D receptor (VDR) for 1,25 (OH)2D3 induction of SIRT1 gene expression.
(F) Effect of VDR knock-down on SIRT1 protein content evaluated by confocal imaging of ShControl and ShVDR HCT 116 colon cancer cells to detect SIRT1 (green). Scale bars represent 25 μm. On the right, quantification of fluorescence intensity using ImageJ software; for each experiment, 3 different fields were evaluated per slide.
(G) Western blot analysis of the effect of 1,25 (OH)2D3 on nuclear accumulation of SIRT1 in cells depleted (ShVDR) or not (ShControl) of VDR.
(H) Specificity of 1,25 (OH)2D3 effects on SIRT1 by western blot analysis of the alternative nuclear sirtuin, SIRT7.
Statistical analysis of 3 independent experiments in each panel was performed by Student t-test (A) to (D) and (H) or by One-Way ANOVA (E)-(F). For (G), statistical analysis of the four groups by ANOVA is represented by * and comparison of the two indicated groups by Students t test, by #. In all panels, values represent mean ± SEM of triplicates corresponding to biological replicates; * or # p< 0.05; ** p< 0.01; *** p< 0.001.