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. Author manuscript; available in PMC: 2012 Nov 15.
Published in final edited form as: Cancer Cell. 2011 Nov 15;20(5):620–634. doi: 10.1016/j.ccr.2011.10.001

Figure 5. CDK4/6 Stabilize FOXM1 by Multisite Phosphorylation.

Figure 5

(A) U2OS and melanoma cells (red) were incubated with DMSO or 1 μM PD0332991 (PD) for 16 h, and endogenous FOXM1 protein levels were analyzed.

(B) U2OS cells stably expressing either control (sh-con) or RB1 targeting shRNA (sh-RB1) were treated with PD0332991 and endogenous FOXM1 was analyzed.

(C) U2OS cells were transfected with pBABE empty vector (EV) or pBABE-FOXM1 and treated with PD0332991.

(D) Cells were co-transfected with either pBABE empty vector (EV) or pBABE-FOXM1, and cyclin D1-CDK4 (14) or cyclin D3-CDK6 (36). Expression of the HA-tagged cyclins is shown below.

(E) U2OS cells transfected with pBABE-FOXM1 were treated with 5 μM MG132 (MG) 4 h prior to cell lysis.

(F) U2OS cells were transfected with pBABE-FOXM1 and either control (si-con) or CDH1 siRNA (si-CDH1).

(G) Effect of replacing the FOXM1 N-terminal (S4/S35), 12 TAD or all 14 CDK (12 TAD plus N-terminal) sites with aspartic acid residues on FOXM1 protein levels. U2OS cells were transfected with either pBABE wild-type FOXM1 (WT), FOXM1 S4/S35 to D (S4D/S35D), ‘12D’ or ‘14D’ mutant. Cells were treated with DMSO (−) or 1μM PD0332991 (PD) for 16 h. See also Figure S3.