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. 2023 Apr 18;4(4):101015. doi: 10.1016/j.xcrm.2023.101015

Figure 2.

Figure 2

Validation of CK1α as a target to treat ENZA-resistant PCa

(A) In vitro proliferation of 22Rv1 cells expressing sgCtrl or two sgRNAs against CK1α was determined by AquaBluer assay after treatment for 72 h with the indicated concentrations of ENZA. Inset: immunoblot (IB) analysis of whole-cell lysates (WCLs) of 22Rv1 cells expressing sgCtrl or two sgRNAs against CK1α.

(B and C) C4-2R (B) or MR49F (C) cells expressing short hairpin control (shCtrl) or two shRNAs targeting CK1α were pre-treated with 100 ng mL−1 doxycycline (Dox) for 48 h to induce CK1α knockdown and then treated with ENZA for 72 h, followed by AquaBluer assay to determine cell viability. Insets: IB analysis of WCL of C4-2R (B) or MR49F (C) cells expressing shCtrl or shCK1α treated with 100 ng mL−1 Dox for 48 h.

(D) 22Rv1 cells expressing sgCtrl or sgRNA targeting CK1α, treated with different concentrations (10, 20, 40, and 80 μM) of ENZA for 24 h and subjected to IB.

(E) IB analysis of WCL derived from C4-2 cells stably expressing empty vector (EV) or FLAG-CK1α after treatment with different concentrations (10, 20, 40, and 80 μM) of ENZA for 24 h.

(F–H) In vivo 22Rv1 xenograft assay. (F) A schema showing the experiment design of in vivo 22Rv1 xenograft and treatment strategy. 22Rv1 (sgCtrl or sgCK1α#1) cells were injected into the flanks of pre-castrated male nude mice. When the tumors reached 100 mm3, the mice (n = 5 for sgCtrl, n = 6 for sgCK1α#1) were treated with vehicle or ENZA (20 mg kg−1 by oral gavage daily, 5 days on and 2 days off for 5 weeks). Tumor volume (G) and tumor weight (H) are shown as mean ± SEM.

(I–K) In vivo LuCaP 35CR PDX assay. (I) A schema showing the experimental design of in vivo LuCaP 35CR PDX. LuCaP 35CR tumor bits were implanted into the flanks of pre-castrated NSG mice. When the tumors reached 50 mm3, the mice were pre-treated with ENZA (50 mg kg−1, by oral gavage daily, 5 days on and 2 days off for 1 week) and then treated with vehicle (n = 11) or BTX-A51 (5 mg kg−1, by oral gavage, two days on and one day off, n = 10) or ENZA (20 mg kg−1, by oral gavage every 3 days, n = 11) or the combination (n = 10) for the indicated time. Tumor volume (J) is shown as mean ± SEM, and survival percentage is shown in (K).