The expression of Orai1, STIM1 and TRPC1 does not change during CytD and CalA treatment in both LNCaP and NED-LNCaP.

<p>(<b>A</b>) Real-time quantitative PCR for the Orai1 transcripts in both LNCaP and NED-LNCaP cells treated with 50 nM CalA for 10 min or 5 µM Cyt D for 10 min. (<b>B</b>) Real-time quantitative PCR for the STIM1 transcripts in both LNCaP and NED-LNCaP cells treated with 50 nM CalA for 10 min or 5 µM Cyt D for 10 min. (<b>C</b>) Real-time quantitative PCR for the TRPC1 transcripts in both LNCaP and NED-LNCaP cells treated with 50 nM CalA for 10 min or 5 µM Cyt D for 10 min. There was no statistical significance observed in each condition (LNCaP and NED-LNCaP). n = 3, done in triplicates.</p

F-actin polymerization by calyculin A (CalA) in both control and NED-LNCaP cells.

<p>(<b>A</b>) Representative images of immunofluorescence staining of F-actin with phalloidin-FITC in NED- and NED-CalA (50 nM for 10 min)-LNCaP cells. n = 3. (<b>B</b>) Histograms representing Tg-induced SOCE quantification (Tg 1 µM, 2 mM extracellular Ca²⁺) in CT-, CT-CalA-, NED- and NED-CalA-LNCaP cells. n = 3, N = 30–40 cells, in triplicates. Asterisks denote statistical significance: * p<0.05.</p

Cytochalasine D (CytD) partially restores the store-operated current induced by TG.

<p>(<b>A</b>) The whole-cell electrophysiological recordings of SOCE induced by 1 µM TG in LNCaP cells control (CT), NED (NED), and NED LNCaP cells treated with 3 µM CytD (NED-CytD). (<b>B</b>) Histograms showing the current density quantifications in the above conditions, n = 3, N = 23; in triplicates, ** - p<0.01.</p

Regulation of Ca²⁺ homeostasis and F-actin polymerization in NED-LNCaP cells.

<p>After 4 (<b>A</b>) or one (<b>B</b>) days of culture in a charcoal-stripped culture medium used to induce NED, the capacitative Ca²⁺ entry quantified by Ca²⁺ imaging is induced by the application of 1 µM Tg in the presence of 2 mM extracellulaire CaCl_2. Asterisks denote statistical significance * - p<0.05; ** - p<0.01, n = 3, N = 30–40 cells, in triplicates. The presence of F-actin fibres was observed in both CT-LNCaP (<b>C</b>) and NED-LNCaP (<b>D</b>) using phalloidin-FITC. Scale bar equals 10 µM. n = 3. <b>E</b>, Expression of Orai1, STIM1, Orai2, and Orai3 as compared to β-Actin in LNCaP cells and NED-LNCaP cells using semiquantitative PCR. n = 2. <b>F</b>, Quantitative real-time PCR for Orai1 and STIM1 in LNCaP (CT) versus LNCaP-NED (NED), n = 3, * - P<0.05.</p

Primers and siRNA.

<p>Primers and siRNA.</p

The effects of 1,25-dihydroxyvitamin D3 on androgen-independent cell lines.

<p><b>A</b>, <b>B</b>, The effects of 1,25-dihydroxyvitamin D3 on androgen receptor-deficient DU-145 cell line in both 2 and 10% FCS-containing RPMI medium (<b>A</b> and <b>B</b>, respectively), * - P<0.05 (as compared to control), n = 3. <b>C, D</b>, The effects of 1,25-dihydroxyvitamin D3 on androgen-insensitive LNCaP C4-2 cell line in both 2 and 10% FCS-containing RPMI medium (<b>C</b> and <b>D</b>, respectively), * - P<0.05 (as compared to control), n = 3. <b>E</b>, the relative expression levels of TRPV6 channel in DU-145 cells treated with 100 µM 1,25-dihydroxyvitamin D3 for 3 days in 2 and 10% FCS-containing RPMI medium, * - P<0.05 (as compared to control), n = 3. <b>F</b>, the expression of TRPV6 channel induced by 100 nM 1,25-dihydroxyvitamin D3 for 3 days in LNCaP cells in steroid-deprived RPMI medium (LNCaP-ST), n = 3.</p

The effects of 1,25-dihydroxyvitamin D3 on proliferation and apoptosis resistance of LNCaP cells are mediated via TRPV6 channel.

<p><b>A</b>, LNCaP cells proliferation in 2% FCS-containing RPMI medium treated with 1,25-dihydroxyvitamin D3 (100 nM, applied at D1), siRNA-TRPV6 (siTRPV6, 80 nM, transfected at D0), the combined treatment of 1,25-dihydroxyvitamin D3 and siTRPV6 specified above, and siRNA-AR (siAR, 80 nM, transfected at D0) as a positive control. * - P<0.05, ** - P<0.01, as compared to control, n = 4; <b>B</b>, a cell cycle assay of LNCaP cells (incubated with 2% FCS-containing RPMI medium) for the same conditions as in MTS assay (<b>A</b>) (D3 equals 100 nM 1,25-dihydroxyvitamin D3), carried out by flow cytometry of the cells stained with propidium iodide. * - P<0.05, ** - P<0.01, § - P<0.05 vs. Vitamin D3; n = 3. <b>C</b>, a western-blotting of proliferating cell nuclear antigen (PCNA) in the conditions indicated above as compared to β-actin. <b>D</b>, an apoptosis assay carried out by flow cytometry as a subG1 population of LNCaP cells cultured in 2% FCS-containing RPMI medium stained with propidium iodide. * - P<0.01 vs. control; n = 3.</p