Fig. 2
RepID-dependent CUL4 recruitment on chromatin is required for cell cycle progression. a siRNAs for CUL4A, CUL4B or CUL4A + B were transfected into RepID WT and KO U2OS cells. After 5 days, cells were labeled by EdU for 30 min, collected and analyzed by flow cytometry. Percentages of cells in each cell cycle phase are indicated (upper panel). Graphs depicting the distributions of G1, S and G2/M phase cells in control vs. CUL4A + B siRNA transfected cells are shown in the bottom panel. Error bars represent standard deviations from three independent experiments. b Chromatin-bound proteins were isolated from cells collected as in a and analyzed by immunoblotting. The numbers under the panels represent the intensity ratios compared to the intensity of the signal in control-siRNA transfected RepID WT cells. c CUL4A + B-siRNA transfected RepID WT and KO U2OS cells were labeled with EdU for 30 min, and immunofluorescence analysis was performed using CUL4A, γH2AX, phospho-RPA, or PCNA antibodies after pre-extraction (upper and middle panel). Scale bar indicates 10 μm. The intensity of each protein was determined in G1 (EdU negative) and ES (EdU positive) using the Pearson’s correlation coefficient. p values were calculated using a two-tailed t-test (n = 10) (bottom panel). d Mitotic HCT116 RepID WT or KO cells were collected after released from a nocodazole block, reseeded in fresh medium and harvested at the indicated time points after incubation with EdU for 30 min. Cells were then analyzed by flow cytometry, and the percentage of cells in each cell cycle phase is indicated as in a. e Aliquots of cells in d were analyzed by immunoblotting after extraction of chromatin fractions. f Graphs representing the percentages of cells in G1, G2/M phase and the ratio of cells in S phase vs. G1 at the indicated time points from three independent experiments. g Schematic of the expected cell cycle patterns in relation to the abundance of chromatin-bound CUL4 (High to Low) in RepID WT, RepID KO, and CUL4A + B-siRNA transfected RepID KO cells
