Figure 2
Figure 2. shRNA knockdown of UBE2D3 suppresses ATRA-induced cell growth arrest. (A) Colony-forming assay in soft agar. Uninfected, control shRNA-infected, and UBE2D3 shRNA-infected NB4 cells were seeded in soft-agar plates in the presence (0.5 μM) or absence of ATRA. The expression of shRNA by the infected cells was confirmed by their coexpression of GFP. The figure shows the results of a representative experiment. Pictures at day 3 and day 7 are presented. Images were viewed using an Olympus 20×/0.5 NA dry objective microscope (Olympus, Melville, NY) and SensiCam CD camera (Cooke, Romulus, MI) and processed using IPlab imaging software (IPlab, Rockville, MD). (B) shRNA knockdown of UBE2D3 suppresses ATRA-induced cell growth arrest in liquid cultures. Control shRNA and UBE2D3 shRNA-infected NB4 cells were cultured in liquid medium containing 0.5 μM ATRA. At each indicated time point, the total number of cells was counted by hemocytometer. The percentages of transduced cells (n %) were analyzed for GFP expression by flow cytometry. The total number of infected cells equals the total amount of cells times n %. Bars indicate 1 standard deviation (SD) about the mean (n = 6, * P < .01). (C) Confirmation of the ATRA resistance by a liquid culturing assay. NB4 cells were infected with UBE2D3 shRNA viruses. The infection efficiency is 10% to 15% under our experimental condition. The infected cells (including 10%-15% infected and 85%-90% uninfected cells) were continuously cultured in liquid medium with or without 0.5 μM ATRA. The percentages of transduced (GFP positive) cells were analyzed by flow cytometry every day. The increase of this number indicates a growth advantage of these cells over the uninfected cells. Data presented are the means (± SD) of 6 independent experiments. (D) shRNA knockdown of UBE2D3 does not promote NB4 cell growth in the absences of ATRA. Cell proliferation was analyzed as described in (B). Data presented are the means (± SD) of 6 independent experiments. (E) shRNA knockdown of UBE2D3 does not suppress staurosporine (STS)-induced cell growth arrest. Control shRNA and UBE2D3 shRNA-infected NB4 cells were cultured in the presence of 0.5 μM staurosporine and the cell growth was assessed as described in (B). Data presented are the means (± SD) of 3 independent experiments.

shRNA knockdown of UBE2D3 suppresses ATRA-induced cell growth arrest. (A) Colony-forming assay in soft agar. Uninfected, control shRNA-infected, and UBE2D3 shRNA-infected NB4 cells were seeded in soft-agar plates in the presence (0.5 μM) or absence of ATRA. The expression of shRNA by the infected cells was confirmed by their coexpression of GFP. The figure shows the results of a representative experiment. Pictures at day 3 and day 7 are presented. Images were viewed using an Olympus 20×/0.5 NA dry objective microscope (Olympus, Melville, NY) and SensiCam CD camera (Cooke, Romulus, MI) and processed using IPlab imaging software (IPlab, Rockville, MD). (B) shRNA knockdown of UBE2D3 suppresses ATRA-induced cell growth arrest in liquid cultures. Control shRNA and UBE2D3 shRNA-infected NB4 cells were cultured in liquid medium containing 0.5 μM ATRA. At each indicated time point, the total number of cells was counted by hemocytometer. The percentages of transduced cells (n %) were analyzed for GFP expression by flow cytometry. The total number of infected cells equals the total amount of cells times n %. Bars indicate 1 standard deviation (SD) about the mean (n = 6, * P < .01). (C) Confirmation of the ATRA resistance by a liquid culturing assay. NB4 cells were infected with UBE2D3 shRNA viruses. The infection efficiency is 10% to 15% under our experimental condition. The infected cells (including 10%-15% infected and 85%-90% uninfected cells) were continuously cultured in liquid medium with or without 0.5 μM ATRA. The percentages of transduced (GFP positive) cells were analyzed by flow cytometry every day. The increase of this number indicates a growth advantage of these cells over the uninfected cells. Data presented are the means (± SD) of 6 independent experiments. (D) shRNA knockdown of UBE2D3 does not promote NB4 cell growth in the absences of ATRA. Cell proliferation was analyzed as described in (B). Data presented are the means (± SD) of 6 independent experiments. (E) shRNA knockdown of UBE2D3 does not suppress staurosporine (STS)-induced cell growth arrest. Control shRNA and UBE2D3 shRNA-infected NB4 cells were cultured in the presence of 0.5 μM staurosporine and the cell growth was assessed as described in (B). Data presented are the means (± SD) of 3 independent experiments.

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