Stable expression of SMRTβ in the ATRA-resistant NB4-MRA1 APL cells promotes ligand binding, ligand-induced transcriptional activation, and ATRA-induced cell differentiation. (A) Effects of stable expression of SMRTβ isoform on the ATRA-binding capacity of PML/RARα mutant I410T in A1-GFP-SMRTβ APL cells. SMRTβ induces ATRA binding to PML/RARα I410T in A1-GFP-SMRTβ cells (▾) compared with A1-GFP cells (•). Nuclear extracts were incubated with 10 nM [³H]-ATRA or with the addition of 200-fold excess unlabeled ATRA and subjected to HPLC analysis usinga6HR 10/30 size exclusion column. Nuclear extracts of stably transfected cells A1-GFP and A1-GFP-SMRTβ were subjected to Western blot analysis for the SMRTα (top band) and SMRTβ (bottom band). (B) Effects of stable SMRTβ expression on transcriptional activation. Cells stably expressing the empty GFP vector (A1-GFP) or the GFP vector harboring SMRTβ (A1-GFP-SMRTβ) were transiently transfected with the RARE reporter gene DR5-tk-CAT. Assays were performed in triplicate and repeated at least 3 times. Bars represent standard deviation of the mean. There was a significant difference between cells stably transfected with SMRTβ compared with transfected cells with the empty vector: ^*P < .05, ^**P < .0005. Nuclear extracts of stably transfected cells A1-GFP and A1-GFP-SMRTβ were subjected to Western blot analysis for the 110-kDa PML/RARα mutant I410T (arrow). (C-D) Expression of the cell-surface myeloid-specific differentiation markers CD11b (C) and CD11c (D) by flow cytometry analysis. Cells stably expressing the empty GFP vector (A1-GFP) or the GFP vector harboring SMRTβ (A1-GFP-SMRTβ) were treated with 0.01 and 1 μM ATRA for 5 days, stained with an antibody specific for CD11b or CD11c, and subjected to flow cytometry analysis. The cells were also analyzed for the isotype control PE-conjugated mouse IgG1κ for CD11b-PE and CD11c-PE. In each sample, viable cells were gated, and expression of CD11b or CD11c surface markers of 5 × 10³ cells was evaluated. Numbers in parentheses indicate the percentage of positive CD11b or CD11c cells. This experiment is representative of 3 that gave comparable results.