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. Author manuscript; available in PMC: 2017 May 11.
Published in final edited form as: Cancer Res. 2016 Mar 28;76(8):2301–2313. doi: 10.1158/0008-5472.CAN-15-0728

Figure 5.

Figure 5

Acquisition of resistance to CDK4/6 inhibition through RB1 loss or cyclin E1 amplification. A, BrdUrd incorporation-ELISA assay in parental (MCF-7, T47D) and palbociclib-resistant (MCF-7pR and T47DpR) cells treated with vehicle or palbociclib for 72 hours (*, P < 0.001 vehicle vs. palbociclib; NS, not significant, two-way ANOVA with Sidak multiple comparisons test). B, cells treated for 96 hours with vehicle or 500 nmol/L palbociclib, and lysates blotted with the indicated antibodies. C, comparison of copy number for MCF-7 parental versus MCF-7pR–resistant cell line. There is an amplification of chromosome 19q12 region encompassing the CCNE1 gene. D, CCNE1 relative copy number change assessment by ddPCR against two different reference genes, RNase P and TERT. E, BrdUrd incorporation-ELISA assay in cells transfected 4 days earlier with control siCON2 or SMARTpool siRNA targeting CDK4 or cyclin D1. Western blot analysis of the same experiment showing knockdowns in MCF-7 cells. F, Western blots of MCF-7 and MCF-7pR cells treated for 24, 48, and 72 hours with palbociclib (Palbo) and blotted phospho-CDK2 Thr160 and total CDK2. G and H, cells transfected for 4 days with control siCON2 or the indicated SMARTpool siRNAs and treated with vehicle or palbociclib for 72 hours. G, BrdUrd incorporation-ELISA assay adjusted with viable cell number. H, cell number after vehicle or palbociclib for 72 hours (**, P < 0.001; ***, P < 0.0001, two-way ANOVA with Tukey multiple comparisons test).