Maintenance of leukemogenic capacity of CML cells by MgkL cell-derived TGF-β1. (A-B) Total, CD41⁺CD150⁺ MgkL, or CD16/CD32⁺ myeloid-lineage cells were purified from BCR-ABL⁺KLS⁺-derived colonies on day 9 and incubated for 24 hours to obtain culture supernatants. The supernatants were subjected to enzyme-linked immunoassay for TGF-β1 (A) or added to the culture of total BCR-ABL⁺ (B) cells to determine the MFIs of intracellular phosphorylated Smad2/3 levels 2 hours later (n = 4). (C) Schematic representation of the experimental procedure of serial colony plating. WT- or DKO-derived BCR-ABL–transduced KLS⁺ cells were cultured under colony-forming conditions in the presence or absence of a neutralizing anti-TGF-β antibody for 7 days. Ten thousand cells obtained from each colony were subjected to the secondary colony formation assay. (D) Colony formation in the secondary plating. The colonies were counted on day 7 (n = 6). Data are presented as the mean ± SD. (E) Survival of secondary transplantation recipients. WT mice were treated with daily administration of galunisertib or DMSO on days 10 through 16 after the initiation of the primary transplantation. Then, bone marrow cells were obtained from the recipients 17 days after the transplantation. For secondary transplantation, the resultant 5 × 10⁴ lineage⁻c-kit⁺ cells were transplanted to other mice, whose survival rates were evaluated (n = 6). Statistical significance was evaluated using the 1-way ANOVA with Tukey’s multiple-comparisons test. Survival rates were compared using the log-rank test. **P < .01.