BACH2 deletion contributes to increased colony-formation abilities and increases cell adhesion. (A) Representative colonies of BACH2^KO and BACH2^Con MCL cells in MethoCult medium were photographed under a microscope. Scale bar, 100 μm. (B) BACH2^KO or BACH2^Con SP53 MCL cells (3.5 × 10⁶) were subcutaneously (s.c.) injected into NOD/SCID mice (n = 10). Mice 1 to 5 were injected with BACH2^Con cells, and mice 6 to 10 were injected with BACH2^KO cells. Xenografts were sacrificed 4 weeks postinjection. Dotted circles represent the area of subcutaneous tumors (top). Tumors were isolated and photographed against an inch ruler (bottom). (C) The size of the tumors in each group was measured. The results are shown as the mean ± SD. **P < .01 (vs control xenografts; Student t test). (D) Human leukocyte cells were isolated from the spleen (SP) and bone marrow (BM) using anti–human CD45-MicroBeads, and the number of CD45⁺ cells was counted using trypan blue staining. The percentage of CD45⁺ cells in each organ was calculated, and data are presented as the mean ± SD. **P < .01 (vs control xenografts; Student t test). (E) BACH2^KO SP53 cells or control cells (3 × 10⁶) were IV injected (i.v.) into NOD/SCID mice (n = 8). Mice 11 to 14 were injected with BACH2^Con cells, and mice 15 to 18 were injected with BACH2^KO cells. Xenografts were sacrificed 8 weeks postinjection. SP and BM were collected and stained for human leukocyte cells using anti–human CD45 antibody. Samples were then analyzed for CD45⁺ cells using FACS analysis. The percentage of CD45⁺ cells in each organ was calculated, and data are presented as the mean ± SD. **P < .01 (vs control xenografts; Student t test). (F) Representative microscopic images of adherent MCL cells in a coculture setting. Either BACH2^KO or BACH2^Con MCL cells were stained with PKH26 prior to seeding onto a preestablished monolayer of HS5 BMSCs. Scale bar, 100 μm. The arrows point to the representative MCL cells adhered to bone marrow stromal cells. (G) CXCR4 levels in BACH2^KO or BACH2^Con cells were analyzed by flow cytometry, the percentage of CXCR4⁺ cells in the population is indicated. (H) BACH2^KO or BACH2^Con MCL cells were serum starved for 48 hours. IL-6 levels were measured using enzyme-linked immunosorbent assay. The colorimetric values in the BACH2^KO cells were normalized to the control values. The results are shown as the mean ± SD from 3 independent experiments. **P < .01 (vs control cells; Student t test).