The role of promoter methylation and PRDM1β expression in PRDM1α inhibition. (A) Schematic diagram showing PRDM1α and PRDM1β promoter CpG island (gray horizontal bar). CpG-dinucleotides (vertical lines), amplification primers (forward and reverse arrows) and sequencing (seq) primer at PRDM1α and PRDM1β promoters are depicted. The broken arrow indicates start of transcription. (B) Bisulfite pyrosequencing analysis of PRDM1α and PRDM1β promoter in cHL cell lines and normal tonsillar CD19⁺ B cells. The methylation intensity is shown by color coding. (C) Bisulfite pyrosequencing analysis of PRDM1α promoter in microdissected HRS cells of 9 cHL cases. (D) PRDM1β is upregulated in cHL cell lines. End point RT-PCR analysis of PRDM1α and PRDM1β expression in cHL and MM cell lines using PRDM1α and PRDM1β specific primers. GAPDH was used as a reference gene. (E) PRDM1β/PRDM1α ratio in cHL and MM cell lines were measured by TaqMan Q-RT-PCR using PRDM1α and PRDM1β specific primers. (F-G) PRDM1β has reduced toxicity to cHL cell lines as compared with PRDM1α. cHL cell lines were transduced with lentiviral SF-LV-cDNA-EGFP vector expressing PRDM1β or EV. (F) PRDM1β protein expression in transduced cell lines is shown by immunoblot. (G) Percentage of live GFP⁺ cells was monitored over 9 days by flow cytometry as described in Figure 4A. Data are presented as mean of 2 independent experiments.