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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 1.

Figure 1

Early adaptive resistance limits the efficacy of CDK4/6 inhibition. A and B, MCF-7 cells treated with 500 nmol/L palbociclib for 24 or 72 hours as indicated followed by 2 hours exposure to BrdUrd. A, images show BrdUrd-positive cells (green) and BrdUrd-negative cells (red). Graphs show number of nuclei (DAPI staining), percentage of BrdUrd–positive cells, and cell area (***, P < 0.001, one-way ANOVA with Tukey multiple comparisons test). B, BrdUrd staining (green) and tubulin (red) illustrate that BrdUrd-negative cells correlate with extended cell area (senescence like phenotype; *, P < 0.05, Student t test). C and D, Western blot analysis of lysates from MCF-7 cells treated for 24 to 72 hours with palbociclib (Palbo). *, addition of fresh vehicle or drug every 24 hours over a 72-hour period. E, MCF-7 cells treated with palbociclib for 72 hours followed by 0.5 hours exposure to EdU, then washed and exposed to BrdUrd for another 24 hours. Graph shows percentage of BrdUrd-positive–EdU-negative cells. F, effect of the drug library on the relative sensitivity to palbociclib expressed as a z score, with z scores below −2 indicating increased sensitivity to palbociclib. Main hits are indicated with arrows. H, clonogenic survival assays in MCF-7 cells treated continuously for 14 days with vehicle, 500 nmol/L palbociclib, 250 nmol/L GDC-0941 (0941; PI3K inhibitor), or 100 nmol/L MK2206 (AKT inhibitor) or 100 nmol/L everolimus (Ever; mTOR inhibitor), and the indicated combinations with palbociclib. I, BrdUrd incorporation measured by ELISA and corrected for viable cell number. Cells treated with vehicle, palbociclib, GDC-0941, or the combination for 24 or 72 hours (*, P < 0.05 palbociclib vs. combination at 72 hours, two-way ANOVA with Tukey multiple comparisons test; **, P < 0.01 palbociclib 24 hours vs. 72 hours; ***, P < 0.001 GDC0941 24 hours vs. 72 hours, two-way ANOVA with Bonferroni posttest).