Figure 1
CDK4/6 activation is tightly coupled with Rb phosphorylation and E2F1 mRNA induction. (A) Schematics of CDK4/6, CDK2 (left), and APC/CCdh1 (right) reporter. Active CDK4/6 and CDK2 phosphorylate and induce translocation of reporters to the cytoplasm. As APC/CCdh1 is inactivated, intensity of the reporter starts increasing. (B) Top, representative images of CDK4/6, CDK2, and APC/CCdh1 reporters, phosphorylation of Rb (p-Rb, S807/811) immunostaining, and mRNA FISH for E2F1 mRNA in the same MCF-10A cells. Scale bar is 20 µm. Bottom, histograms of CDK4/6, CDK2, and APC/CCdh1 activities and p-Rb at S807/811. Red dotted line indicates a threshold used to classify active CDK4/6 and CDK2, inactive APC/CCdh1, and p-Rb populations. After 48 h mitogen removal, cells were stimulated with mitogens for 14 h prior to fixation. (C) Scatter plot of the percentage of cells with active CDK4/6 and CDK2, inactive APC/CCdh1, p-Rb at S807/811, and averaged E2F1 mRNA levels in MCF-10A cells as a function of time after mitogen stimulation. Solid line represents sigmoidal best-fit line. (D) Single-cell correlation of p-Rb at S807/811 versus CDK 4/6 activity (top) and CDK2 activity (bottom) in MCF-10A cells at various time points after mitogen stimulation (8, 10, 12 and 14 h). Cell density is color-coded. (E) Three-dimensional activity map of CDK4/6 versus CDK2 activity in MCF-10A cells where cell density, the percentage of inactivated APC/CCdh1, the percentage of p-Rb at S807/811, and E2F1 mRNA levels are color-coded. (F,G) Representative images of multiplexed staining for p-Rb (S807/811, T373, S608, S780) in MCF-10A cells (F). Scale bar is 20 µm. Three-dimensional activity map of CDK4/6 versus CDK2 activity where cell density, the percentage of inactivated APC/CCdh1, and the percentage of p-Rb at S807/811, T373, S608, or S780 are color-coded (G). After 48 h mitogen removal, MCF-10A cells were stimulated with mitogens for 12 h prior to fixation.
