Figure 2
CDK4/6 activation induces Rb hyperphosphorylation and inactivation. (A,B) Immunoblot of p-Rb (S807/811), total Rb, and β-actin in MCF-10A cells at various time points after mitogen stimulation. After 48 h mitogen removal, MCF-10A cells were stimulated with mitogens for indicated time and then treated with DMSO, palbociclib (1 µM), or palbociclib (1 µM) + CDK1/2 inhibitor III (1 µM) for 15 min prior to harvesting cells (B). (C) Three-dimensional activity maps of CDK4/6 versus CDK2 activity where cell density, the percentage of inactivated APC/CCdh1, and the percentage of p-Rb at sites S807/811 are color-coded. After 48 h mitogen removal, MCF-10A cells were stimulated with mitogens for 12 h and treated with DMSO, roscovitine (60 µM), CDK2 inhibitor III (60 µM), palbociclib (1 µM), abemaciclib (1 µM), or ribociclib (1 µM) for 1 h prior to fixation. (D) Left, representative images of nuclear-bound Rb after in situ extraction and classification of nuclear-bound and nuclear-unbound Rb. Scale bar is 20 μm. Right, three-dimensional activity maps of CDK4/6 versus CDK2 with corresponding cell density, the percentage of inactivated APC/CCdh1, and the percentage of nuclear-unbound Rb color-coded (left to right). After 48 h mitogen removal, MCF-10A cells were stimulated with mitogens for 12 h prior to in situ extraction and fixation.
