Fig. 2
From: Targeting TRPV6/CXCR4 complexes prevents castration-resistant prostate cancer metastasis to the bone
TRPV6 is involved in cancer cell migration and invasion in vitro. a Basal migration of PC-3Mtrpv6−/−-mCherry, PC-3Mtrpv6−/−-pTRPV6wt, and PC-3Mtrpv6−/−-pTRPV6D582A stable cell clones. Representative images and quantification of migrated cells (n = 4). Scale bar, 200 μm. b Directed migration of PC-3Mtrpv6−/−-mCherry, PC-3Mtrpv6−/−-pTRPV6wt, and PC-3Mtrpv6−/−-pTRPV6D582A stable cell clones shown as representative images and quantification of migrated cells (n = 4). Scale bar, 200 μm. c Tracking of PC-3Mtrpv6−/−-mCherry (n = 29), PC-3Mtrpv6−/−-pTRPV6wt (n = 46), and PC-3Mtrpv6−/−-pTRPV6D582A (n = 30) stable cell clones for 48 h. d Quantification of both the cell velocity and distance of stable cell clones from (c). e PC-3Mtrpv6−/− (n = 63) and PC-3Mtrpv6+/+ (n = 115) cells were tracked for 48 h. f Quantification of both the cell velocity and distance of prostate cancer cells from (e). g Tracking of PC-3Mtrpv6+/+ cells treated with 40 nM of either the negative control siRNA (n = 86) or a mixture of siRNAs against TRPV6 (n = 46) for 48 h. h Quantification of both the cell velocity and distance of prostate cancer cells from (g). i Basal invasion of PC-3Mtrpv6−/−-mCherry, PC-3Mtrpv6−/−-pTRPV6wt, and PC-3Mtrpv6−/−-pTRPV6D582A stable cell clones shown as representative images and quantification of migrated cells (n = 4). Scale bar, 200 μm. j Directed invasion of PC-3Mtrpv6−/−-mCherry, PC-3Mtrpv6−/−-pTRPV6wt, and PC-3Mtrpv6−/−-pTRPV6D582A stable cell clones shown as representative images and quantification of migrated cells (n = 4). Scale bar, 200 μm. k Basal and direct invasion of PC-3Mtrpv6−/−-pTRPV6wt cells treated with 40 nM control (siNC) or specific siTRPV6. Quantification of invasive cells (n = 4). l Flow cytometry analysis of cell surface TRPV6 expression in PC-3Mtrpv6−/−-mCherry (n = 29), PC-3Mtrpv6−/−-pTRPV6wt (n = 46), and PC-3Mtrpv6−/−-pTRPV6D582A stable cell clones. m TRPV6 channel targeting the plasma membrane in the regions of cell protrusions in PC-3Mtrpv6−/−-mCherry, PC-3Mtrpv6−/−-pTRPV6wt, and PC-3Mtrpv6−/−-pTRPV6D582A stable cell clones. Scale bars, 100 μm. n EMT marker and transcription factor expression via qPCR in PC-3Mtrpv6−/−-mCherry, PC-3Mtrpv6−/−-pTRPV6wt, and PC-3Mtrpv6−/−-pTRPV6D582A stable cell clones (n = 3). o Western blot analysis of N-cadherin and calpain-2 proteins expression in PC-3Mtrpv6−/− and PC-3M-luc-C6trpv6+/+ stable cell clones compared with TRPV6 expression. p Vimentin staining in PC-3Mtrpv6−/−-mCherry, PC-3Mtrpv6−/−-pTRPV6wt, and PC-3Mtrpv6−/−-pTRPV6D582A stable cell clones with the quantification of the mean intensity reported via Hoechst staining (n = 6). Scale bar, 20 μm. q N-cadherin staining in PC-3Mtrpv6−/−-mCherry, PC-3Mtrpv6−/−-pTRPV6wt, and PC-3Mtrpv6−/−-pTRPV6D582A stable cell clones with the quantification of the mean intensity reported via Hoechst staining (n = 6). Scale bar, 20 μm. r E-cadherin staining in PC-3Mtrpv6−/−-mCherry, PC-3Mtrpv6−/−-pTRPV6wt, and PC-3Mtrpv6−/−-pTRPV6D582A stable cell clones with the quantification of the mean intensity reported via Hoechst staining (n = 6). Scale bar, 20 μm. s MMPs and TIMP2 expression via qPCR in PC-3Mtrpv6−/−-mCherry, PC-3Mtrpv6−/−-pTRPV6wt, and PC-3Mtrpv6−/−-pTRPV6D582A stable cell clones (n = 3). t Protein expression of the MMP2, MMP3, MT-MMP1, and MMP9 proteins in PC-3Mtrpv6-/- and PC-3M-luc-C6trpv6+/+ stable cell clones compared with β-actin expression and TRPV6 status. u 1% gelatin zymography analysis and quantification of MMP2 in the conditioned media of PC-3Mtrpv6−/−-mCherry, PC-3Mtrpv6−/−-pTRPV6wt, and PC-3Mtrpv6−/−-pTRPV6D582A stable cell clones. Mean ± SEM (a, b, d, f, h–l, n, p–s, u). Two-tailed t-test (f, h). Two-way ANOVA (a, b, d, i–l, n, p–s, u). See also Supplementary Figs. 1 and 2
