Figure 2. Abemaciclib inhibited cell proliferation, and led to G1/S arrest and a long-term reduction in phosphorylation of Rb in ER+ breast cancer cell lines.

(A, B) Luminal ER+ breast cancer cells were treated with abemaciclib at 8 different concentrations ranging from 2.22 and 0.001 uM. Inhibition of pRb^S780 (brown line) was measured after 16 hours of treatment by immunofluorescence. Proliferation inhibition (green line) and changes in the cell cycle (red, black, and blue lines) were measured after 4 days (A) and 10 days of treatment (B) by monitoring PI staining. Cell cycle changes are described as the percentage of cells in the 4n subpopulation, (blue), in the 2n subpopulation (red) and in a subpopulation with intermediate DNA content (black). (C) T47-D cells failed to reenter the cell cycle following treatment with abemaciclib. T47-D cells were treated for 1-8 days with 500 nM of abemaciclib (continuous treatment) and then were re-plated at 30–40% confluency and maintained in fresh growth medium without abemaciclib for 3 or 6 additional days (washout). Protein lysates were prepared from the cells and the inhibition of cell cycle reentry was measured by monitoring the inhibition of pRb (S807/S811) and Topo IIα expression by immunoblotting. Images of Western blots were cropped to denote the relevant band(s) for clarity.