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. 2019 Sep 26;2(6):453–467. doi: 10.1021/acsptsci.9b00065

Figure 4.

Figure 4

Sensitivity to ralaniten is dependent upon expression of UGT2B15orUGT2B17. (A) Cells treated with 15 nM siRNA targeting UGT2B15 and UGT2B17 efficiently silenced mRNA expression. Significant cross-reactivity was observed with respect to UGT2B15 and UGT2B17; however, no other isoforms were silenced by siRNA treatment. Data presented as mean ± SEM and analyzed by two-way ANOVA with Dunnett’s test applied post hoc, n = 4 independent experiments. (B) Western blots of UGT2B15/17 protein levels in LNCaP-RALR cells transfected with 15 nM control, UGT2B15 (top), or UGT2B17 (bottom) siRNA for 24–96 h. (C) Crystal violet assay measuring proliferation of cells 96 h post-transfection with either scrambled (SCRM), UGT2B15, and/or UGT2B17 siRNA (15 nM). Twenty-four hours after transfection, cells were treated with ralaniten (25 μM) or DMSO vehicle and stimulated with 1 nM R1881 (black bars) or EtOH (white bars). Silencing of either UGT2B15 or UGT2B17 was sufficient to restore sensitivity to ralaniten (n = 3 independent experiments). Data presented as mean ± SEM and analyzed by two-way ANOVA with Dunnett’s test applied posthoc,n = 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.0001; n.s., not significant; RAL, ralaniten.