Figure 6.

CDK6 Regulates Transcription of the Pro-Angiogenic Factor Vegf-A

(A) Ex vivo VEGF-A protein levels (pg/ml) of the subcutaneous tumors (see Figures 5A–5C) were analyzed with an ELISA experiment (n = 3).

(B) Relative Vegf-A mRNA levels of indicated cells were analyzed by qPCR. The fold change compared to Cdk6^−/−Vegf-A mRNA level is shown (n ≥ 4; Cdk6^−/− versus: Cdk6^−/−+Cdk6, ^∗∗∗p < 0.0001; Cdk6^−/−+Cdk6R31C, ^∗p = 0.03; Cdk6^−/−+Cdk6K43M, ^∗∗p = 0.004).

(C and D) ChIP assays were performed on (C) Cdk6^−/− and Cdk6^−/− cells expressing an HA-tagged CDK6 using different amounts of an anti-HA-antibody as well as on (D) Cdk6^−/− and Cdk6^+/++Cdk6 cells using an anti-CDK6-antibody. PCR was performed to detect the Vegf-A promoter sequence.

(E) Dose-response curve of Cdk6^+/+ p185^BCR-ABL-transformed cells treated 24 hr with the CDK6/4 inhibitor PD0332991 (n = 3).

(F) Vegf-A mRNA levels of Cdk6^+/+ p185^BCR-ABL-transformed cells (n = 3) treated 24 hr with 0, 30, 100, 300, and 1000 nM PD0332991 were analyzed by qPCR. The fold change compared to untreated Vegf-A mRNA levels is shown.

(G) p16^INK4a mRNA levels of Cdk6^+/+ p185^BCR-ABL–transformed cells (n = 3) treated 24 hr with 0, 30, 100, 300, and 1,000 nM PD0332991 were analyzed with qPCR. The fold change compared to untreated p16^INK4a mRNA levels is shown.

Error bars indicate the mean ± SEM. See also Figure S6.