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. 2020 Mar 21;12(3):748. doi: 10.3390/cancers12030748

Figure 1.

Figure 1

Androgen-androgen receptor (AR) signaling upregulates the transcription of KLF5 in PCa cells. (a–d) R1881 induced the expression of KLF5 at both protein (a, c) and RNA (b, d) levels in LNCaP (a, b) and C4-2B (c, d) cells. After 24-hour culture in phenol red–free RPMI-1640 medium containing 10% charcoal-stripped (CS) FBS, cells were treated with R1881 for 24 h at the indicated concentrations. Western blotting and real-time qPCR were performed to detect protein and mRNA respectively. (e,f) R1881 induced the expression of KLF5 at both protein (e) and RNA (f) levels at the indicated times in C4-2B cells. Cell culture conditions and the detection of KLF5 protein and mRNA were the same as in panels a-d. After 24-hour culture, cells were treated with R1881 (10 nM) for the indicated times. (g–j) Enzalutamide inhibited the expression of KLF5 at both protein (g, i) and RNA (h, j) levels in LNCaP (g, h) and C4-2B (i, j) cells. Cells were cultured in complete media for 24 h and treated with enzalutamide at the indicated concentrations for 24 h. (k,l) Enzalutamide inhibited the expression of KLF5 at both protein (k) and RNA (l) levels at the indicated times in C4-2B cells. Cell culture conditions and the detection of KLF5 protein and mRNA were the same as in panels g-j. After 24-hour culture, cells were treated with enzalutamide (Enz, 10 µM) for the indicated times. (m,n) RNAi-mediated silencing of AR prevented R1881 from upregulating KLF5 expression at both protein (m) and mRNA (n) levels in C4-2B cells. Cell culture conditions and the detection of KLF5 protein and mRNA were the same as in panels a-d. Transfection of siRNAs was for 6 h before R1881 treatment (10 nM). Enzalutamide (Enz, 10 µM) was used as a control. siCtrl, control siRNA; siAR, AR siRNA. (o) Knockdown of AR also prevented R1881 from inducing transcriptional activity of the KLF5 promoter in C4-2B cells, as detected by the promoter luciferase reporter activity assay. Experimental conditions were the same as in panels i and j except that the reporter plasmid was co-transfected with siRNAs. ns, not significant; *, p < 0.05; **, p < 0.01; ***p < 0.001.