Protein levels for asynchronous mcf10a cells in g0, g1, s, g2, and m phases of the cell cycle.
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(A) Cell-cycle signaling network depicting in red the proteins measured in this study. (B) Density scatter plot of EdU versus DNA content used to define G0/G1, early S, S, late S, and G2/M populations, as marked by the boxed populations. Note that y-axis units for Fig 1 B–D are in log base 10, such that, for example, a y-axis value of 2.8 = 102.8 = 631; y-axis units are arbitrary fluorescence units. (C) Density scatter plot of phospho-Rb-S807/811 versus DNA content. EdU-negative cells with approximately 2N DNA content can be subdivided into a G0/quiescent population with hypo-phosphorylated Rb and a G1 population with hyper-phosphorylated Rb, as marked by the boxed populations. (D) Density scatter plot of phospho-Histone H3 versus DNA content. EdU-negative cells with approximately 4N DNA content can be subdivided into a G2 population with no phospho-Histone H3 signal and a mitotic population that is phospho-Histone H3-positive, as marked by the boxed population. (E) Top: Cells were tracked by live-cell imaging for 24 hours using H2B-mTurquoise as a nuclear marker, while simultaneously monitoring CDK2 activity using DHB-mVenus. Cells were then fixed and stained with various antibodies against proteins of interest (POI).
Protein levels for asynchronous MCF10A cells in G0, G1, S, G2, and M phases of the cell cycle.
(A–H) Column 1: Density scatter of the indicated protein versus DNA content; data are pooled from 9 IF images from 1 representative well. Column 2: Contour plot of the indicated protein versus DNA content; contours are color-coded by cell-cycle phase according to the legend. Data are pooled from 9 IF images from 1 representative well. Column 3: Histogram (probability density) of the indicated protein for G0 cells (purple, defined as 2N DNA content, EdU-negative, and hypo-phosphorylated Rb) versus G1 cells (blue, defined as 2N DNA content, EdU-negative, and hyper-phosphorylated Rb). Two biological replicates are shown; each replicate represents 9 pooled IF images. Note that the y-axes for Column 1 and Column 2, and the x-axis for Column 3, are in log base 10; units are arbitrary fluorescence units. All signals are nuclear except where indicated. Also note that because of cell rounding during mitosis, IF signal intensities are artificially high in mitotic cells. Abbreviation: Cyto-CycB1, Cytoplasmic Cyclin B1 signal; IF, immunofluorescence; Rb, retinoblastoma protein.