Deletion of IRF4 in miR-125b cells. (A) Analysis of genetic mutations in cancer B cells through comparative genomic hybridization microarray (aCGH). Genomic DNA harvested from sorted miR-125b–overexpressing GFP⁺CD19⁺ cancer B cells was subjected to aCGH analysis. Genomic DNA from normal C57bl/6 mice of the same gender (female) was used as controls. The figure represents the relative amount of genetic content of the cancer sample vs the C57bl/6 control (plotted as ratio in y-axis). A genomic region was considered different between the samples if 3 consecutive probes exhibited different signal intensities in the microarray. Regions highlighted in green and orange indicate genomic areas in which the cancer cells have higher and lower DNA content, respectively. The other aCGH analysis is displayed in supplemental Figure 6A. (B) G-band karyotyping of miR-125b–induced cancer B cells. Trisomy 11 is highlighted in red box. (C) Flow cytometric analysis of cancer B cells. Sorted GFP⁺CD19⁺ cells isolated from independent groups of MG-125b mice were transplanted into recipient mice. Upon cancer development, the bone marrow of these mice (highlight in red) was analyzed by flow cytometry. The plot shows the samples within the CD19⁺ gated population. The sample overlaid in black represents total BMCs harvested from healthy control C57bl/6 mice. Group 1 and group 2 corresponds to the aCGH samples displayed in panel A and supplemental Figure 6A, respectively. (D) IRF4 locus and genotyping primer sequences. IRF4 locus is shown with exons represented as solid bars. “Start,” “ex,” and “int” signifies translation start site, exon, and intron, respectively. The red bars represent the PCR product amplified by the primers used for genotyping in panel E-F and supplemental Figure 6E. (E) Quantitative PCR analysis of IRF4 locus. The relative amount of genomic DNA from miR-125b–induced cancer B cells were quantified by quantitative PCR and normalized to control Hsp70 locus. The control samples are genomic DNA harvested from normal C57bl/6 mice. The PCR amplifies a region spanning the translational start site (denoted as “start” in panel C). (F) Genomic DNA from miR-125b–induced cancer B cells was subjected to genotyping analysis. Exon2 (ex2), exon4 (ex4), and exon6 (ex6) of IRF4 were assessed. Bach1 locus was used as positive control. Samples 3-4 and 5-6 correspond to miR-125b–induced cancer B cells harvested from mice originating from group 1 and group 2 described in Figure 5G, respectively. The agarose gel images of the PCRs are displayed.