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. Author manuscript; available in PMC: 2020 Jul 1.
Published in final edited form as: Mol Cancer Ther. 2019 Sep 23;19(1):231–246. doi: 10.1158/1535-7163.MCT-19-0508

Figure 2. ENZ-liganded androgen receptor induces Jak2 phosphorylation in prostate cancer cells.

Figure 2.

A, CWR22Pc, LAPC4 and ENZ-R cells were cultured with ENZ or Jak2 inhibitor AZD1480 alone or in combination for 12 days at indicated concentrations. Expression levels of active Stat5 were determined by immunoprecipitation (IP) of Stat5 followed by immunoblotting (WB) for pStat5a/b and total Stat5. Whole cell lysates (WCL) were immunoblotted for pStat3, Stat3 and Actin. B, Genetic knockdown of Jak2 blocks ENZ-induced Stat5 phosphorylation in PC cells. Jak2 was suppressed by lentiviral Jak2 shRNA vs. shCtrl in PC cells for 3 days followed by ENZ or vehicle for 7 days. Active Stat5, Stat5 and Jak2 levels were evaluated by IP and WB, as depicted. C, CWR22Pc and LAPC-4 cells were treated with ENZ or vehicle for 6 h at the indicated concentrations. Control cells were stimulated with prolactin (Prl) (10 nM) for 20 min as control for cytokine-induced Jak2 phosphorylation. Alternatively, PC cells were treated with ENZ or vehicle for 12 days or stimulated with Prl for 20 min at the indicated concentrations. Stat5 and Jak2 were IP:ed and immunoblotted (WB) for pStat5, pJak2, total Stat5, and total Jak2. WCLs were immunoblotted for Actin. D, AR is required for ENZ induction of Jak2 activation. AR was suppressed in CWR22Pc cells by lentiviral AR shRNA (shAR) vs. shCtrl for 3 days followed by ENZ for 6 h. In parallel experiments, CWR22Pc cell were cultured with 1.5 μM DHT for 6 h or 7 days. ENZ induction of Jak2-Stat5 activation requires a cytokine receptor shown by treatment of CWR22Pc cells with ENZ alone or in combination with a prolactin receptor antagonist LFA102 for 6 h or 7 days at the indicated concentrations. E, ENZ induction of Jak2-Stat5 activation requires ongoing protein synthesis in PC cells. CWR22Pc cells were treated with cyclohexamide (CHX) (35 μM) for 24 h followed by ENZ treatment (40 μM) for 6 h. F, Phosphatase inhibitor Vanadate (VAN) abolished ENZ induction of Jak2 phosphorylation. CWR22Pc cells were treated with ENZ (6 h) or VAN (2 h) or vehicle alone or pre-treated with VAN (2 h) followed by ENZ (6 h). G, ENZ-induced Jak2 phosphorylation is decreased by depletion of either PTPƐ or SHP-2 phosphatases. CWR22Pc and LAPC4 cells were infected with lentivirus expressing shRNA targeting PTPƐ, SHP-2, PTP1B or shCtrl for 3 days followed by ENZ or vehicle treatment for 6 h at the indicated concentrations. IPs and WBs in were conducted as described in A.