Cyclin D1 is a target of UBE2D3. (A) UBE2D3 physically associates with cyclin D1 in both ATRA-treated (48 hour) and untreated NB4 cells, and ATRA treatment leads to increased ubiquitination of cyclin D1. Cell lysates were immunoprecipitated with cyclin D1 antiserum or IgG. The precipitates were blotted with monoclonal anti-UBE2D3, antiubiquitin, as well as antiactin antibodies. The left two lanes contain small aliquots of the whole-cell lysates used for coimmunoprecipitation. The experiments were repeated multiple times. The figure shows the results of a representative experiment. The levels of ubiquitinated cyclin D1 were quantified using NIH ImageJ software. All samples were compared with the signal detected in untreated NB4 cell lysate immunoprecipitated with anti-cyclin D1 antiserum (lane 3). Data presented are the means (±SD) of 3 independent experiments. *P < .001. (B) Cyclin D1 protein level is down-regulated in ATRA-treated NB4 cells. NB4 cells were cultured in the presence of 0.5 μM ATRA for the indicated time. Cyclin D1 and actin were detected by Western-blotting analysis. The relative amounts of cyclin D1 protein were quantified using NIH ImageJ software. The cyclin D1 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 are presented as mean values from 3 independent experiments whose results varied less than 5%. (C) Cyclin D1 mRNA level in ATRA-treated NB4 cells analyzed by RNA microarray. Cyclin D1 mRNA expression was analyzed using the same microarray data as described in Figure 3B. Both absolute values (left) and fold increases (right) were presented. The IDs of each oligomeric probe are indicated. Data presented are the means (± SD) of 3 independent experiments. (D) Cyclin D1 mRNA level in ATRA-treated NB4 cells analyzed by real-time quantitative PCR. The experiment was conducted as described in Figure 3C. Shown are fold increases over untreated cells. Data presented are the means (±SD) of 3 independent experiments. *P < .01. (E) UBE2D3 shRNA completely abolished ATRA-induced cyclin D1 degradation 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. Protein extracts were resolved on SDS-PAGE and the amounts of cyclin D1 protein were measured as described in panel B. Data presented are the means (± SD) of 3 independent experiments. *P < .001. (F) ATRA-induced cyclin D1 degradation could not be detected in the ATRA-resistant NB4-R2 cells. ATRA treatment and quantification of cyclin D1 protein level by Western blotting were conducted exactly as described in panel B. (G) Knocking down cyclin D1 protein by siRNA inhibits the growth of UBE2D3-shRNA-expressing NB4 cells in the presence of ATRA (abrogates the effect of the UBE2D3 shRNA). NB4 cells stably expressing UBE2D3 shRNA (2 million) were transfected with human cyclin D1 siRNA (100 pmol) (Santa Cruz Biotechnology, Santa Cruz, CA) or control siRNA (100 pmol) using a Nucleofector Kit (Amaxa, Koeln, Germany) and a protocol provided by the manufacturer. Using this electroporation-based method, transfection efficiencies (for siRNA) of greater than 80% are routinely obtained. Transfected NB4 cells were cultured in the presence of 0.5 μM ATRA and the cell growth was assessed as described in Figure 2B. Data presented are the means (± SD) of 3 independent experiments. *P < .01 versus cells transfected with control siRNA. (Inset) Western blot result showing the specific knockdown of cyclin D1 protein. (H) Overexpression of cyclin D1 suppresses ATRA-induced cell growth arrest in NB4 cells (mimics the effect of the UBE2D3 shRNA). NB4 cells (2 ×106) were transfected with a cyclin D1 expression plasmid (6 μg; Open Biosystems, Huntsville, AL) or a pEGFP expression plasmid (control) using the Nucleofector Kit. We routinely obtain transfection efficiencies (for plasmid) of greater than 20% using this method. Transfected NB4 cells were cultured in the presence of 0.5 μM ATRA and the cell growth was assessed as described in Figure 2B. The ATRA-resistant cells gain growth advantage in ATRA-containing medium. Data presented are the means (± SD) of 3 independent experiments. *P < .01 versus cells transfected with control plasmid. (Inset) Western blot result showing the overexpression of cyclin D1 protein in ATRA-treated (3 days) NB4 cells.
