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. 2019 Aug 16;146(6):1686–1699. doi: 10.1002/ijc.32607

Figure 2.

Figure 2

The interplay between AR and MMP9 and PIP5K1α in VCaP cells. (a) Immunoblots show the expression of AR in control (0.1% DMSO) treated or DHT treated VCaP cells expressing either control (pCMV) or AR expressing vectors (pCMV‐AR). Data is presented as average of three independent experiments (mean values of AR expression in control pCMV cells and pCMV‐AR cells was 0.70 and 1.03, difference = 0.33, 95% CI = 1.01–1.06, p < 0.001; Mean AR expression in control pCMV cells and DHT‐treated pCMV cells was 0.70 and 1.04, difference = 0.34, 95% CI = 1.02–1.06, p < 0.001; Mean AR expression in control pCMV cells and in DHT‐treated pCMV‐AR cells was 0.70 and 1.5, difference = 0.80, 95% CI = 1.44–1.55, p < 0.001). (b) Representative immunofluorescent images show the expression and subcellular localization of AR in VCaP cells overexpressing AR or control vector that were treated with DMSO or DHT. (c) Immunoblots show the expression of MMP9 in either control or DHT treated VCaP cells expressing pCMV or pCMV‐AR vectors (p = 0.008 vs. p = 0.03). (d, e). Immunoblots show the expression of PIP5K1α and pAKT S473 in (mean values of PIP5K1α expression in pCMV control cells and in pCMV‐AR cells were = 0.66 and 0.85, difference = 0.19, 95% CI = 0.84–0.85, p < 0.001; Mean PIP5K1α expression in pCMV control cells and in DHT‐treated pCMV cells were = 0.66 and 0.88, difference = 0.22, 95% CI = 0.87–0.88; p = 0.002). PIP5K1α was detected on the same gel/membrane as for AR, and they share the same GAPDH as a loading control. Mean value of pAKT expression in pCMV control cells and in pCMV‐AR cells were 0.85 and 1.15, difference = 0.3, p = 0.009; Mean value of pAKT expression in pCMV control cells and in DHT‐treated pCMV cells were 0.85 and 1.06, difference = 0.21, 95% CI = 1.02–1.09, p = 0.03), and for VEGF, VEGFR1 and VEGFR2 (for VEGF expression, p = 0.002; VEGF expression in DHT‐treated cells, p = 0.023; for VEGFR1, p < 0.001; for VEGFR2, p = 0.024; VEGFR2 in DHT‐treated cells, p = 0.01). Data are presented as average of three experiments. (f) Migration assay shows the effect of induced AR overexpression on the migratory ability of VCaP cells. Data are presented as average of three experiments (mean migrated pCMV control cells = 178, mean migrated pCMV‐AR cells = 286, difference = 108, 95% CI = 257–314, p = 0.004). p < 0.05, as indicated by “*”, p < 0.01, as indicated by “**”. Androgen Receptor (Santa Cruz), MMP9 (ab38898, Abcam), VEGF (Upstate Biotechnology), VEGFR1 and VEGFR2 (Santa Cruz), alpha‐Tubulin (Biosite), pAkt (Ser473, Cell Signaling and Biosite) and PIP5K1α (Protein Technologies) were used.