Figure 5.

CDK6 Regulates Tumor Angiogenesis

(A–C) Cdk6^+/+, Cdk6^−/−, Cdk6^−/−+Cdk6, Cdk6^−/−+Cdk6R31C, or Cdk6^−/−+Cdk6K43M p185^BCR-ABL-transformed cells were injected subcutaneously (sc) into Nu/Nu mice (n = 3 cell lines/genotype; n ≥ 6 tumors/genotype). (A) Immunofluorescence staining for CD31 (red) was performed to analyze blood vessel formation in sc tumors. Original magnification 20×. Representative cases of each genotype are depicted. (B) Quantitative assessment (HistoQuest) of the blood vessels of the subcutaneous tumors (n ≥ 4 tumors of three independent cell lines; Cdk6^−/− versus: Cdk6^+/+, ^∗∗p = 0.001; Cdk6^−/−+Cdk6, ^∗∗∗p < 0.0001; Cdk6^−/−+Cdk6R31C, ^∗∗∗p = 0.0002; Cdk6^−/−+Cdk6 K43M, ^∗∗∗p < 0.0001). (C) Tumor weight was detected after 8 days of injection (Cdk6^−/− versus: Cdk6^+/+, ^∗∗p = 0.003; Cdk6^−/−+Cdk6R31C, ^∗∗∗p = 0.0001).

(D and E) Murine endothelial cell (mEC) spheroids were cultured in methylcellulose with 20% supernatant derived from indicated cells for 24 hr. (D) Quantitative analysis of the relative sprout length was measured with ImageJ software (n ≥ 3; Cdk6^−/− versus: Cdk6^+/+, ^∗p = 0.042; Cdk6^−/−+Cdk6, ^∗∗p = 0.002; Cdk6^−/−+Cdk6R31C, ^∗∗p = 0.009; Cdk6^−/−+Cdk6K43M, ^∗p = 0.038). (E) One representative set of pictures is given.

(F and G) A monolayer wounding assay was performed to analyze migration of mECs incubated with supernatant derived from indicated cells. After 2 and 24 hr, pictures were taken and mEC migration quantified (% open area after 24 hr) with TScratch Software. (F) n ≥ 5; Cdk6^−/− versus: Cdk6^+/+, ^∗∗∗p = 0.0007; Cdk6^−/−+Cdk6, ^∗∗p = 0.0014; Cdk6^−/−+Cdk6R31C, ^∗∗p = 0.0092; Cdk6^−/−+Cdk6K43M, ^∗∗∗p = 0.0009). (G) One representative set of pictures is given.

Error bars indicate the mean ± SEM. See also Figure S5.