Figure 3
Figure 3. UBE2D3 expression is up-regulated in ATRA-treated NB4 cells. (A) NB4 cells were cultured in the presence of 0.5 μM ATRA for the indicated time. Protein extracts were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). UBE2D3 and actin were detected by Western blotting analysis. The relative amounts of UBE2D3 were quantified using NIH ImageJ software (http://rsb.info.nih.gov/ij/). The UBE2D3 signals were normalized to the amount of actin in each sample. All samples were compared with the signal detected in untreated (time point 0) NB4 cells. Data presented are the means (± SD) of 3 independent experiments. (B) ATRA-induced up-regulation of UBE2D3 mRNA expression identified by RNA microarray. The mRNA was prepared from untreated or ATRA-treated (1 μM for 4 days) NB4 cells. Both absolute values (left) and fold increases (right) were presented. The IDs of each oligomeric probe are indicated. All probes showed a significant ATRA-induced augmentation of UBE2D3 mRNA level. Data presented are the means (± SD) of 3 independent experiments. (C) ATRA-induced up-regulation of UBE2D3 mRNA expression identified by real-time quantitative PCR. NB4 cells were cultured in the presence of 1 μM of ATRA for the indicated number of days. The 2-step quantitative reverse transcription-polymerase chain reactions (RT-PCRs) were conducted using purified total RNA as described in Document S1. The expression of alpha tubulin (k-alpha-1) was used as internal control gene. Shown are fold increases over untreated cells. Data presented are the means (± SD) of 3 independent experiments. *P < .01 versus control. (D) ATRA-induced UBE2D3 up-regulation could not be detected in the ATRA-resistant NB4-R2 cells. ATRA treatment and quantification of UBE2D3 protein level were conducted exactly as described in panel A. (E) UBE2D3 shRNA completely abolished ATRA-induced up-regulation of UBE2D3 in NB4 cells. Control shRNA and UBE2D3 shRNA-infected NB4 cells were incubated with or without 0.5 μM ATRA for 6 hours. The GFP-positive shRNA-expressing cells were sorted on a MoFlo High-Performance cell sorter (Dako, Carpinteria, CA). Protein extracts were resolved on SDS-PAGE and the amounts of UBE2D3 protein were measured as described in panel A. Data presented are the means (± SD) of 3 independent experiments. *P < .001

UBE2D3 expression is up-regulated in ATRA-treated NB4 cells. (A) NB4 cells were cultured in the presence of 0.5 μM ATRA for the indicated time. Protein extracts were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). UBE2D3 and actin were detected by Western blotting analysis. The relative amounts of UBE2D3 were quantified using NIH ImageJ software (http://rsb.info.nih.gov/ij/). The UBE2D3 signals were normalized to the amount of actin in each sample. All samples were compared with the signal detected in untreated (time point 0) NB4 cells. Data presented are the means (± SD) of 3 independent experiments. (B) ATRA-induced up-regulation of UBE2D3 mRNA expression identified by RNA microarray. The mRNA was prepared from untreated or ATRA-treated (1 μM for 4 days) NB4 cells. Both absolute values (left) and fold increases (right) were presented. The IDs of each oligomeric probe are indicated. All probes showed a significant ATRA-induced augmentation of UBE2D3 mRNA level. Data presented are the means (± SD) of 3 independent experiments. (C) ATRA-induced up-regulation of UBE2D3 mRNA expression identified by real-time quantitative PCR. NB4 cells were cultured in the presence of 1 μM of ATRA for the indicated number of days. The 2-step quantitative reverse transcription-polymerase chain reactions (RT-PCRs) were conducted using purified total RNA as described in Document S1. The expression of alpha tubulin (k-alpha-1) was used as internal control gene. Shown are fold increases over untreated cells. Data presented are the means (± SD) of 3 independent experiments. *P < .01 versus control. (D) ATRA-induced UBE2D3 up-regulation could not be detected in the ATRA-resistant NB4-R2 cells. ATRA treatment and quantification of UBE2D3 protein level were conducted exactly as described in panel A. (E) UBE2D3 shRNA completely abolished ATRA-induced up-regulation of UBE2D3 in NB4 cells. Control shRNA and UBE2D3 shRNA-infected NB4 cells were incubated with or without 0.5 μM ATRA for 6 hours. The GFP-positive shRNA-expressing cells were sorted on a MoFlo High-Performance cell sorter (Dako, Carpinteria, CA). Protein extracts were resolved on SDS-PAGE and the amounts of UBE2D3 protein were measured as described in panel A. Data presented are the means (± SD) of 3 independent experiments. *P < .001

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