Figure 1. Experimental workflow and positive controls.
(A) NB4 cells were differentially treated using serum starvation (SS), hydroxyurea (HU), and RO-3306 to arrest cells in G0/G1, S, and G2 phases of the cell cycle, respectively. Cells were then processed for label-free quantitative MS-based proteomics in a similar manner to the previous analysis of elutriated cells (Ly et al., 2014). (B) Asynchronous and arrested cells were stained with a DNA-binding dye and analyzed by flow cytometry. DNA content histograms are shown. Cells with >4N DNA content (∼3.3%) are highlighted in the RO-3306 data. (C) Immunoblot analysis of the arrested cells using antibodies recognizing cell cycle phase-specific markers (cyclin A, cyclin E, and cyclin B1) and beta tubulin as a loading control. (D) Pairwise comparisons of MS-based protein abundances (LFQ intensities) independently processed and measured from three asynchronous NB4 cultures. Pearson correlation coefficients are reported in the top left corner of each scatter plot.