Immunohistochemical analyses.

<p>Representative immunohistochemical figures of ductal infiltrating breast cancer with low VDR expression (score 3) (a) and with high VDR expression (score 8) (b). Scale bar 30 μ. (c) The MCA graph demonstrated that phenotype AA is located in the quadrant containing the most aggressive parameters (T3 tumours, HER2 positive, ER negative and G3 in contrast to phenotype GG which is associated to more favorable bio-pathological parameters [T1 tumours, N0, G1, ki67 (-)].</p

<i>Cdx2</i> status in human breast cancer cell lines by ER status.

<p>^aEstrogen receptor.</p><p><i>Cdx2</i> status in human breast cancer cell lines by ER status.</p

Biopathological characteristics of the 80 Breast Cancer patients.

<p>Biopathological characteristics of the 80 Breast Cancer patients.</p

Relationship between polymorphism <i>Cdx2</i> and VDR expression in 80 BC cases and according to molecular subtype.

<p>VDR is differently expressed in <i>Cdx2</i> phenotype (a). There is no difference in VDR expression according to <i>Cdx2</i> phenotype in LA (b) and LB (c). In contrast, in HS (d) and in TN (e) BC, VDR is more likely to be expressed in GA and GG phenotypes.</p

VDR transcript and protein basal levels.

<p>(a, c) Q-PCR for the expression of VDR gene from a panel of breast cancer cell lines. (b, d) Representative western blot analysis of whole cell lysates obtained from breast cancer cell lines stained with the indicated antibodies. GAPDH staining was used as a loading control. Cells were separated based on their <i>Cdx2</i> status. (e) Q-PCR for the expression of CYP24A1 gene from eight representative breast cancer cell lines treated or not with 100 nM of 1,25(OH)2D₃ for 24 hours.</p

Association between polymorphism Cdx2 and ER status.

<p>P-value (Chi-Square test) < 0.0001.</p><p>Association between polymorphism Cdx2 and ER status.</p

Vitamin D anticancer effects in vitro on ER(–) breast cell lines with GG <i>Cdx2</i> status.

<p>Time-dependent growth curves of BT20 (a) and HCC1954 (b) cells in the absence or in the presence of different doses of 1,25(OH)2D₃ (25–200 nM). Histograms (c and d) showing average colony percentage over vehicle from duplicate experiments at 10–21 days from seeding. Bars indicate the average of three independent experiments. Representative micrographs of colonies formed by BT-20 (e) and HCC1954 (g) cells treated with 1,25(OH)₂D₃ as indicated. Representative western blot analysis of whole cell lysates obtained from BT20 (e) and HCC1954 (f) cells treated as suggested and stained with the indicated antibodies. GAPDH staining was used as a loading control.</p

Association between polymorphism Cdx2 and Molecular subtypes.

<p>P-value (Chi-Square test) = 0.05.</p><p>Association between polymorphism Cdx2 and Molecular subtypes.</p

Vitamin D wound healing assay.

<p>Upper part: representative micrographs of wound healing closure assays from BT20 (a), SUM159PT (b) and SK-BR3 (c) cells, treated as indicated for 48–72 hrs. Lower part: histogram showing the healing closure efficiency of the cells treated with vehicle or 25 nM 1,25(OH)2D₃ at the indicated times. Bars indicate the average of three independent experiments. * p-value < 0.05.</p

Vitamin D anticancer effects in vitro on ER(–) breast cell lines with AG <i>Cdx2</i> status.

<p>Time-dependent growth curves of SUM159PT (a) and MDA-MB-231 (b) cells in the absence or in the presence of different doses of 1,25(OH)2D₃ (25–200 nM). Histograms (c and d) showing average colony percentage over vehicle from duplicate experiments at 10–21 days from seeding. Bars indicate the average of three independent experiments. Representative micrographs of colonies formed by BT-20 (e) and HCC1954 (g) cells treated with 1,25(OH)₂D₃ as indicated. Representative western blot analysis of whole cell lysates obtained from SUM159PT (e) and MDA-MB-231 (f) cells treated as suggested and stained with the indicated antibodies. GAPDH staining was used as a loading control.</p