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. Author manuscript; available in PMC: 2019 Apr 5.
Published in final edited form as: Cell Rep. 2019 Mar 5;26(10):2667–2680.e7. doi: 10.1016/j.celrep.2019.02.023

Figure 3. CDK6 Expression and Activity Contributes to Cell Survival after Palbociclib Exposure.

Figure 3.

(A) Western blot analysis of shRNA-mediated knockdown of cell-cycle proteins in T47D cells.

(B) Clonogenic survival assay after 24 h of palbociclib exposure on shRNA-expressing T47D cells. Data are reported as the means ± SEMs of 3 independent experiments.

(C) Confirmatory CDK6 knockdown and clonogenic survival assay with an additional CDK6 shRNA. Survival was significantly decreased in both shCDK6-1 and -2 compared to shNT (p < 0.0001). Data are reported as the means ± SEMs of 3 independent experiments.

(D) Western blot of T47D cells with CRISPR/Cas9 knockout of CDK6 using an sgRNA targeting the 5′ UTR, followed by ectopic expression of CDK6 mutants. Clonogenic survival assay of CRISPR/Cas9 knockout CDK6, and mutant add-back lines treated with escalating dose of palbociclib.

(E) Cell-cycle analysis of shCDK6 T47D cells ± 100 nM palbociclib treatment for 24 h. Data are reported as the means ± SEMs of 3 independent experiments. ****p < 0.0001.

(F) Annexin V apoptosis assay using shSCR, shCDK6, and sgCDK6 T47D cells treated with escalating doses of palbociclib for 48 h. Data are reported as the means ± SDs of 2 independent experiments.