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. Author manuscript; available in PMC: 2018 Jun 27.
Published in final edited form as: Clin Cancer Res. 2013 Aug 13;19(20):5658–5674. doi: 10.1158/1078-0432.CCR-13-0422

Figure 1. AZD1480 inhibits Stat5a/b activation and downstream molecular events in prostate cancer (PC) cells. (A) AZD1480 disrupts ligand-induced and constitutive phosphorylation of Stat5a/b in PC cells.

Figure 1

Figure 1

Figure 1

Stat5a and Stat5b were immunoprecipitated (IP) using anti-Stat5a or anti-Stat5b pAbs from (i) CWR22Rv1 and (ii) CWR22Pc cells which had been serum-starved (0% FBS) overnight, pre-treated with AZD1480 at indicated concentrations for 1 h followed by stimulation of the cells with 10 nM human prolactin (hPrl) for 20 min and immunoblotted using anti-phospho-Stat5a/b (anti-pYStat5a/b). Filters were stripped and re-blotted with anti-Stat5a/b mAb. Similarly, Stat5a and Stat5b were IP from exponentially growing (iii) CWR22Rv1 and (iv) CWR22Pc cells and IB for pYStat5a/b and total Stat5a/b. (B) AZD1480 disrupts ligand-induced dimerization of Stat5a/b in PC cells. pCMV-3Flag-Stat5a, pCMV-3Myc-Stat5a and pPrl-receptor (PrlR) plasmids were co-transfected into PC-3 cells. Cells were serum-starved for 16 h, pre-treated with AZD1480 or vehicle (DMSO) at the indicated concentrations for 2 h, followed by stimulation with 10 nM hPrl for 20 minutes. 3Myc-Stat5a was immunoprecipitated with anti-Myc mAb and blotted with anti-Flag mAb or anti-Myc mAb, as indicated. Whole cell lysates were blotted with anti-Myc, anti-Flag mAb or anti-actin pAb to demonstrate the input. (C) AZD1480 inhibits nuclear translocation of ligand-activated Stat5a/b in PC cells. PC-3 cells were transfected with plasmids expressing Stat5a (pStat5a-Flag) and human PrlR (pPrlR). After serum starvation for 16 h, the cells were treated with AZD1480 for 2 h followed by stimulation with 10 nM hPrl for 20 min. Immunostaining of Stat5a/b is demonstrated by indirect immunofluoresence (green), while DAPI staining (blue) shows the nuclei. (D) (i) AZD1480 inhibits binding of Stat5 to DNA, shown by EMSA analysis using the Prl-response element of the beta-casein gene as the probe. COS-7 cells were transiently co-transfected with pStat5a/b and pPrlR, serum-starved for 10 h and pre-treated for 2 h with AZD1480 or vehicle at indicated concentrations followed by stimulation with 10 nM hPrl for 30 min. Nuclear extracts were prepared for EMSA and the specificity of the Stat5-DNA binding complex was demonstrated by supershift with anti-Stat5a pAb vs. normal rabbit serum (NRS). (ii) AZD140 inhibits transcriptional activity of Stat5a/b in PC cells. PC-3 cells were transiently co-transfected with a genomic ß-casein-promoter-luciferase plasmid, pRL-TK (Renilla luciferase), pPrlR, pStat5a or pStat5b, as indicated. Cells were serum-starved for 20 h and pre-treated with AZD1480 at indicated concentrations for 1 h followed by stimulation with hPrl (10 nM) for 16 h. The relative luciferase activities were determined, and the mean values of three independent experiments performed in triplicates +/− SE values are indicated by bars.