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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 3. ENZ induces a formation of a positive Jak2-Stat5 feed-forward loop in prostate cancer.

Figure 3.

A, ENZ induced Jak2 phosphorylation is accompanied by an increase in Stat5 phosphorylation and Jak2 protein levels in PC cells. CWR22Pc, LAPC-4 and ENZ-R cells were treated with ENZ or vehicle during a time-course at the indicated concentrations. PC cells were stimulated with Prl (10 nM) as control for Jak2 and Stat5 phosphorylation. The phosphorylation status of Stat5 and Jak2 were determined by IP of Stat5 and Jak2 followed by immunoblotting for pStat5, total Stat5, pJak2 and total Jak2. WCLs were immunoblotted for Actin. B, ENZ induces Jak2 mRNA levels in PC cells. CWR22Pc and LAPC-4 cells were treated with ENZ or vehicle for indicated periods of time or infected with lentiviral Jak2 shRNA (shJak2) vs. shCtrl (Jak2 mRNA control) (3 days) followed by qRT-PCR. C, Jak2, but not Jak1, levels are induced by ENZ. PC cells were treated with ENZ or vehicle for 12 days. Jak1 and Jak2 were immunoprecipitated and blotted for Jak1 and Jak2. WCLs were immunoblotted for Actin. D, ENZ increases Jak2 protein levels by Stat5-driven up-regulation of Jak2 mRNA expression in PC cells. When Stat5 was depleted in PC cells by lentiviral Stat5 shRNA (shStat5) or shCtrl for 3 days followed by ENZ or vehicle for 7 days, ENZ failed to increase Jak2 mRNA levels. For Jak2 mRNA control, Jak2 was suppressed by lentiviral Jak2 shRNA (shJak2) or shCtrl (left panel) for 3 days. Jak2 mRNA levels were determined by qPCR and protein levels by IP and WB as described in A. E, CWR22Pc and LAPC-4 cells were infected with lentiviral Stat5 shRNA (shStat5), shCtrl (upper and bottom panels), constitutively active (CA) Stat5 or GFP for 3 days (middle panel). After 3 days, PC cells were treated with Prl for 3 days (bottom panel). IP, WB and qPCR were conducted as described in A and B. F, Schematic representation of the proposed ENZ induction of hyperactivated Jak2-Stat5 feed-forward loop in PC.