Effect of VEN on the expression of BBC3 in B-cell lymphoma cell lines and primary CLL cells. (A) Schematic representation of MeDip-seq results. (B) Schematic drawing of BBC3 promoter region. For the Dual-Glo Luciferase Assay (Figure 2E), a 917 bp big region containing the CpGs of interest was cloned in a CpG-free vector, followed by the luciferase reporter. (C-D) Methylation changes detected by pyrosequencing for the CpG of interest in cell lines (C) and primary CLL cells before and after VEN resistance (D). (E) Dual-Glo Luciferase Assay analysis of methylated (meth) and unmethylated (unmeth) versions of the promoter region of BBC3. Mean ± SD, N = 10. ∗∗∗∗P < .0001, compared with unmethylated reporter construct, Student t test. (F) Methylation of BBC3 promoter region in 5′AZA-treated (5 passages) VEN-resistant B-cell lymphoma cell lines, determined by pyrosequencing. (G) Immunoblot for PUMA in 3 VEN-sensitive and -resistant B-cell lymphoma lines treated with or without 5′AZA (0.1 μM) for 5 passages. (H) Viability assay of 3 VEN-treated cell lines (24 hours, 1 μM) after incubation with 5′AZA (5 passages). Mean ± SD of 3 independent experiments, viability determined by flow cytometry. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001, compared with untreated (-5-AZA) cells, Student t test. (I) Schematic analysis of OCR analysis. (J-K) Mitochondrial respiration and glycolysis in PUMA-KO KARPAS-422 (J) and OSU (K) cells upon injection of the Seahorse Mito Stress test drugs. Data are shown as floating bars (min. to max.) and are representative of 3 to 6 independent experiments. Paired 2-tailed Student t test: ∗P < .05; ∗∗P < .01. ECAR, extracellular acidification rate; KO, knockout; w/o, without.