Adrenomedullin increases PC-3 and T24/83 cell adhesion, migration and invasion.

<p>(A) RT-PCR experiment showing RAMP2, RAMP3 and CLR expression in PC-3 and T24/83 cells. (B) PC-3 (left panel) and T24/83 (right panel) cell adhesion was examined by seeding 3*10⁴ and 1.5*10⁴ cells respectively per well in 96-well plates pre-coated with fibronectin, and incubated for 45 min with or without AM (200 nM) (N = 3, *P<0.05 compared with control cells). β1 integrin phosphorylation was studied by western-blotting on total proteins extracted from PC-3 and T24/83 cells seeded on fibronectin coated plates and treated with or without AM. (C) PC-3 and T24/83 cell migration was studied by Transwell assay after 8 h of treatment (N = 3, *P<0.05 compared with control cells). FAK phosphorylation was studied by western-blotting on total proteins extracted from PC-3 and T24/83 cells treated with or without AM. (D) For invasion assay, transwell membrane was pre-coated with 50 µg Matrigel, and PC-3 and T24/83 cells were let to invade for 24 h (N = 3, *P<0.05 compared with control cells).</p

Adrenomedullin induces TRPV2 translocation to plasma membrane.

<p>(A) The effect of AM (200 nM, 45 min) and TRPV2 silencing (siTRPV2, 50 nM, 48 h) on basal cytosolic calcium of PC-3 and T24-83 cells was studied by calcium imaging. (n = 120 cells, N = 4, *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM). (B) TRPV2 presence at the plasma membrane was examined by biotinylation on T24/83 cells control or either treated with AM (200 nM, 45 min) or AM and PI3K inhibitor LY294.002 (10 µM, added 5 min before AM). (C) Effect of LY294.002 on PC-3 and T24/83 cell migration examined by transwell assay after 8 h incubation with or without AM (N = 3. *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM). (D) Effect of LY294.002 on AM-induced migration of PC-3 and T24/83 TRPV2-silenced cells examined by transwell assay after 8 h incubation with or without AM (N = 3. *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM).</p

Adrenomedullin effect is mediated by TRPV2.

<p>(A) Western-blotting analysis of TRPV2 protein level in PC-3 and T24/83 cells treated with either siCTL or siTRPV2 (50 nM, 48 h). Effect of TRPV2 silencing (siTRPV2, 50 nM, 48 h) (B) on PC-3 and T24/83 cell adhesion to fibroncectin incubated or not with AM (200 nM, 45 min) (N = 3, *P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM); (C) on PC-3 and T24/83 cell migration examined by transwell assay after 8 h incubation with or without AM (N = 3 *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM); (D) on PC-3 and T24/83 cell invasion through matrigel (AM 200 nM, 24 h) (N = 3. *, P<0.05 compared with control cells; ^#P<0.05 compared with control cells treated with AM).</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