shRNA knockdown of UBE2D3 suppresses ATRA-induced cell-cycle arrest. (A) Cell-cycle analysis of ATRA-treated NB4 cells. NB4 cells were cultured in the presence of 0.5 μM ATRA for the indicated time. Cells were stained with propidium iodide and the cellular DNA content, which was reflected by PI staining, was analyzed by FACS using the Cell Quest software (Becton Dickinson). The percentage of cells in each cell-cycle phase was determined using a ModFIT software (Becton Dickinson), which deconvolves the flow-cytometer data. At least 10 000 cells were analyzed for each data point. (B) The cell percentages obtained from panel A were plotted against times of ATRA treatment. Data presented are the means (± SD) of 3 independent experiments. (C) ATRA treatment does not alter cell-cycle distribution in ATRA-resistant NB4-R2 cells. ATRA treatment and cell-cycle analysis were conducted exactly as described in panel A. (D) The proportions of cells that are in each cell-cycle phase were compared between ATRA-treated NB4 cells and NB4-R2 cells. Data presented are the means (± SD) of 3 independent experiments P < .01 versus untreated cells. (E) shRNA knockdown of UBE2D3 suppresses ATRA-induced G0/G1 arrest in NB4 cells. UBE2D3 shRNA-infected NB4 cells were incubated in the presence of 0.5 μM ATRA for 48 hours. Viral-infected and uninfected cells were gated by their high and low fluorescent intensity, respectively. Cell-cycle analysis was carried out as described in panel A. Data are presented as mean values from 3 independent experiments whose results varied less than 5%.