Figure 5.

Basal CDK1 activity is required for AR-regulated locus transactivation. (A) LNCaP cells in CDS medium were treated for 2 h with CDK1 inhibitor RO-3306 (RO, 10 μM), CDK9 inhibitor (iII, 50 μM) and AR antagonist enzalutamide (ENZ, 10 μM). Total proteins were normalized for blotting. (B and C) LNCaP cells in CDS medium were pre-treated for 2 h with CDK1 inhibitor RO-3306 (0.5, 1, 2.5, 5 and 10 μM), followed by 4 h treatment with DHT (10 nM) as indicated. Total proteins were harvested for blotting (B) and total RNA was harvested for qRT-PCR (C) analyses, respectively. (D and E) LNCaP cells in CDS medium were pre-treated for 2 h with indicated inhibitors (RO-3306: RO, 10 μM; Roscovitine: Ros, 10 μM; CGP74514A: CGP, 10 μM; Olomoucine: Olo, 10 μM; PF: Pfizer compound AG024322, 10 μM; Dinaciclib: Dina, 1 μM; CDK1/2 inhibitor: 1/2, 20 μM; and CDK1/5 inhibitor: 1/5, 100 μM), followed by 4 h treatment with DHT (10 nM) as indicated. Total proteins were harvested for blotting (D) and total RNA was harvested for qRT-PCR analysis (E). (F–H) LNCaP cells in CDS medium were transfected with control siRNA or two independent CDK1 siRNAs (20 nM) for 3 days, followed by 4 h treatment with DHT (10 nM). Total proteins were normalized and assessed by immunblotting (F); cells were harvested for ChIP-qPCR analysis (G); or qRT-PCR analysis was carried out with genes of interest (normalized to the DHT negative samples being set as 1) (H). (I and J) LNCaP cells in CDS medium were pre-treated with CDK1 inhibitor RO-3306 (10 μM) for indicated time points, followed by 4 h treatment without or with DHT (10 nM) as indicated. Total proteins were harvested for blotting (F), or cells were subjected to ChIP for AR and pS81 AR (G).