Fig 1: Basic experimental approach. (A) Flow cytometric analysis of U2OS cells pre-extracted with salt and detergent before fixation (extracted) or fixed without pre-extraction (unextracted). Cells were stained with antibodies against MCM3 (MCM) and RB1 (RB1) and the DNA-stain Hoechst (DNA). Density scatter plots are shown for MCM staining versus DNA content (top) and RB1 staining versus DNA content (bottom). (B) and (C) (i) Gating for single cells based on the area (FL2A) and the width (FL2W) of the signal for DNA staining. (ii) Gate defining G1-phase (left), regions defining MCM-positive cells (middle) and cells with RB1 anchored (right). (iii) Scatter plot of MCM versus RB1 for the gated G1 cell population with quadrants indicating the MCM- and RB1-positive and negative cells defined as in B (left), A schematic explanation of quadrants (right). In (C) the samples were stained with secondary antibodies only.
Fig 2: Validation of the method in BJ cells released from G0-phase. (A) Immunoblot analysis of BJ cells released from G0-phase after synchronization by contact inhibition. Cells were pre-extracted with salt and detergent (extracted) or left unextracted (unextracted) before cell lysis and processed for immunoblotting with antibodies against total RB1 (RB1), phosphorylated RB1 on Ser795 (pRB1-Ser795) and γ-tubulin. RB1-pp indicates a hyper-phoshorylated form of RB1 detected by the RB1 antibody. Time points indicate hours after release from G0-phase. Note that the blot shown for pRB-Ser795 was from a separate gel than the others, and the corresponding γ-tubulin blot is shown in Supplementary Figure S1A. (B) BJ cells were synchronized and released as in (A) and analyzed by flow cytometry as in Figure 1B, at the indicated times after release. Lines in the density scatter plots (top and middle rows) indicate regions defining positive and negative cells for either MCM or RB1. Gates for G1-cells are indicated in the DNA histograms (bottom). (C) Scatter plots of MCM versus RB1 for the gated G1 cell population with quadrants indicating the MCM- and RB1-positive and negative cells defined as in (B). Numbers indicate the percentages of G1 cells in each quadrant. (D) Column charts showing the percentages of MCM-positive G1 cells in the scatter plots shown in (C) (quadrants Q1 and Q2). Black bars (Q2) indicate cells with RB1 anchored (before restriction point) and gray bars (Q1) cells with RB1 hyper-phosphorylated and no longer anchored (after restriction point). Note that cells start passing the restriction point from 9 hours (RB1-pp in A and two RB1 populations in (C) from 9 h) and S-phase cells are visible from about 15 h (S-phase pattern of MCM in the 15 h MCM plot in (B)). See Supplementary Figure S1B for EdU incorporation versus MCM loading from a similar experiment.
Fig 3: RB1 hyper-phosphorylation and MCM loading after ionizing radiation. (A) Immunoblot analysis of BJ cells synchronized and released as in Figure 2A and either treated (+) or not treated (−) with 6 Gy X-ray irradiation at 1 h after release. Cells were processed for immunoblotting similar to the unextracted samples in Figure 2A, with antibodies against total RB1 (RB1) and γ-tubulin. (B) BJ cells were synchronized and treated as in (A) and analyzed by flow cytometry as in Figure 2B, except that bar-coding with Pacific Blue was included to minimize sample-to-sample variation. Two non-irradiated and two irradiated samples (e.g. 10 and 13 h) were bar-coded with different concentrations of Pacific Blue and mixed before antibody staining and analysis, allowing the use of identical MCM and RB1 regions for the four samples (Supplementary Figure S1A). The individual samples were thereafter separated by gating on the Pacific Blue-specific signal (Supplementary Figure S1B). (C) Scatter plots of MCM versus RB1 for the gated G1 populations in (B). (D) Column charts showing the percentages of MCM-positive cells in the scatter plots shown in (B), similar as in Figure 2D. Mean values from three independent experiments are shown. Error bars represent standard error of the mean.
Fig 4: CPP30‐Lipo/CDKACT4 reports the CDK4 inhibitor pharmacodynamics in living cells. (A) Western blot of pRb (S795) protein levels from MCF‐7 cells treated with different concentrations of palbociclib. (B) Distribution of MCF‐7 cells treated with different concentrations of palbociclib in different phases of the cell cycle (n = 3). (C, D) Fluorescence images (C) and MFI (D) of MCF‐7 cells treated with different concentrations of palbociclib after incubation with CPP30‐Lipo/CDKACT4 (n = 3, **p < 0.01). (E, F) Representative fields under fluorescence microscopy (E) and MFI (F) of MCF‐7 cells treated with different concentrations of palbociclib after incubation with CPP30‐Lipo/CDKACT4 (n = 3, ***p < 0.001). Scale bar = 20 μm
Fig 5: CPP30‐Lipo/CDKACT4 reports the cell cycle and CDK4 activity in living cells. (A) Timelapse micrographs of dividing cells: overlaid images of phase contrast and spectral representation of relative CPP30‐Lipo/CDKACT4 fluorescence intensity. Scale bar = 10 μm. (B) Western blot of CDK4 and pRb (S795) protein levels from MCF‐7 cells treated with siRNA directed against CDK4 (siCDK4) or none (siNC). (C) Cell distribution of siNC‐treated MCF‐7 cells and siCDK4‐treated MCF‐7 cells in different phases of the cell cycle (n = 3, ***p < 0.001). (D, E) Fluorescence images (D) and mean fluorescence intensity (MFI) (E) of siNC‐treated MCF‐7 cells and siCDK4‐treated MCF‐7 cells after incubation with CPP30‐Lipo/CDKACT4 (n = 3, **p < 0.01). (F, G) Representative fields under fluorescence microscopy (F) and MFI (G) of siNC‐treated MCF‐7 cells and siCDK4‐treated MCF‐7 cells after incubation with CPP30‐Lipo/CDKACT4 (n = 3, ***p < 0.001). Scale bar = 20 μm
Supplier Page from Abcam for Anti-Rb (phospho S795) antibody