Identification of homozygous deletion and promoter methylation of BIM. (A) Array CGH and genomic PCR studies of MCL cell lines, showing interstitial homozygous deletion of BIM gene. In none of the cases was deletion of the BUB1 gene observed. (B) Western blot analysis shows absence of expression of BIM protein in MCL cell lines (in the figure, IRM2 and JVM2) but also in Burkitt lymphoma cell lines (in the figure, Seraphina). (C) Methylation of BIM promoter was frequently detected in Burkitt lymphoma cell lines (in the figure, KHM10B and CA46) and patient biopsies (P22191 and P26267) as well as in a subset of B-NHL from different origins (in the figure, Karpas 1106 and SCI-1) but not in MCL cell lines (in the figure, JVM2 and HBL2) or patient biopsies (P21058 and P21274). (D) BIM promoter methylation was correlated with loss of expression at the RNA and protein levels, and treatment with demethylating Aza restored BIM expression. Error bars indicate standard deviation. (E) A detailed mapping of CpG-methylation of the promoter by bisulfite genomic sequencing confirmed previous data. Samples analyzed correspond to a positive methylated control (C+); peripheral blood (PB) from a healthy donor (unmethylation); the OZ cell line (unmethylated); the Burkitt-derived cell line Namalwa (methylated); and a lymph-node biopsy from a patient with Burkitt lymphoma (P26267) showing a mixture of methylated and unmethylated colonies.