*2.1. Androgen*/*AR Signaling Upregulates KLF5 Transcription in PCa cells*

To test the role of androgen/AR signaling in KLF5 transcription, we measured KLF5 expression in two androgen-responsive PCa cell lines, LNCaP and C4-2B, in hormone-free medium (RIPA1640 medium supplemented with charcoal-stripped bovine fetal serum) treated with varying concentrations of R1881, a synthetic androgen, specifically binds to AR with higher affinity than dihydrotesterone (DHT), and R1881-bound AR dimerizes and translocates to the nucleus to interact with coregulators to regulate gene transcription [34,35]. Androgen treatment caused a dose-dependent increase in KLF5 expression at both protein and mRNA levels (Figure 1a–d). As expected, R1881 treatment also increased the expression of known AR targets *PSA*, *TMPRSS2*, and *FKBP5* at the mRNA level (Figure 1b). Treatment of C4-2B cells with R1881 at 10 nM for different times increased KLF5 expression in a time-dependent manner (Figure 1e,f).

The same two cell lines grown in normal medium, which contains hormones to activate AR signaling, were treated with enzalutamide to block androgen/AR signaling. Enzalutamide is an AR antagonist that binds with AR to block its nuclear translocation and subsequent interactions with its coactivators in regulation target gene transcription [36,37]. Enzalutamide is widely used in the treatment of PCa [38,39]. Enzalutamide treatment at varying concentrations caused a dose-dependent decrease in KLF5 expression at both protein and mRNA levels in both cell lines (Figure 1g–j), while decreasing the expression of AR and its target genes (*PSA*, *TMPRSS2*, and *FKBP5*) as expected (Figure 1h). Consistently, treatment of C4-2B cells with 10 μM enzalutamide for different times decreased the expression of both KLF5 and AR in a time-dependent manner (Figure 1k,l).

To test whether R1881-induced KLF5 expression depends on AR, we knocked down AR by siRNA in C4-2B cells cultured in hormone-free medium in the presence of R1881 (10 nM) or enzalutamide (10 μM) and analyzed KLF5 expression. AR silencing by siRNA, which was confirmed by western blotting (Figure 1m), eliminated the induction of KLF5 by R1881 at both protein and mRNA levels (Figure 1m,n). Although cells were cultured in hormone-free medium, enzalutamide treatment still reduced AR protein (Figure 1m) and KLF5 mRNA levels (Figure 1n). Further supporting a role of AR in R1881-induced KLF5 expression, the promoter-luciferase reporter assay demonstrated that, in C4-2B cells cultured in hormone-free media, R1881 induced a significant KLF5 promoter activity while enzalutamide decreased the activity, and AR silencing eliminated these effects (Figure 1o). These findings indicate that androgen/AR signaling upregulates KLF5 transcription.

**Figure 1.** *Cont.*

**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.
