Defective expression of TβRII was associated with promoter methylation in B-cell lymphoma cell lines. (A) RT-PCR analysis of TβRII transcription in DB and RL cells. Total RNAs (2 μg) from DB and RL cells were used for cDNA synthesis using a primer pair corresponding to full-length cDNA. (B) RT-PCR analysis of TβRII transcription in B-cell lymphoma cell lines before and after 5′-azacytidine treatment. (C) Methylation status of TβRII gene promoter in B-cell lymphoma cell lines before and after 5′-azacytidine treatment. Methylation-specific PCR (MSP) and unmethylation-specific PCR (USP) corresponding to −25 and −140 promoter regions were performed using bisulphate-modified DNA as template. (D) Status of phospho-Smad2 in DB and Akata cells after 5′-azacytidine. An equal amount of whole-cell lysates (60 μg) from DB and Akata cells before and after 5′-azacytidine treatment were analyzed by Western blot analysis. The membranes were stripped and reprobed with total-Smad2 as indicated above. (E) Treatment with 5′-azacytidine rendered DB cells partially responsive to TGF-β. Lymphoma cells were treated with 5′-azacytidine and PMA as described in Materials and methods. After 1 week, cells were plated at 0.1 × 10⁶/mL and treated with TGF-β (10 ng/mL) for various time periods. At the end of each time point, cell counts were performed.