Forced expression of PBX3 coding region exhibits opposite functions than miR-181a/miR-181b. (A) Forced expression of PBX3-CDS could reverse the effects of miR-181a/miR-181b on viability (left panel) and apoptosis (right panel) of MONOMAC-6, THP-1, and KOCL-48 cells, respectively. (B) Cell growth/proliferation analysis of MONOMAC-6 cells transfected with MSCV-PIG (ie, control), MSCV-PIG-miR-181a, MSCV-PIG-miR-181b, MSCV-PIG-PBX3 (CDS), MSCV-PIG-PBX3 (CDS)–miR181a, or MSCV-PIG-PBX3 (CDS)–miR181b. *P < .05. (C) Western blot assay of expression of PBX3 at the protein level in MONOMAC-6 cells 96 hours after transfection with more than 6 individual constructs. (D) Apoptosis analysis by flow cytometry with anti–annexin V antibody staining in MONOMAC-6 cells 72 hours after transfection with more than 6 individual constructs. PI indicates propidium iodide. Red rectangle indicates apoptotic cells. (E) In secondary BMT assay, mice with MA9 + miR181b (n = 10) developed leukemia significantly (P = .02) slower than those of MA9 alone (n = 5). Mice with MA9 + miR181b + PBX3-CDS (n = 6) developed leukemia at a similar speed as those with MA9 + PBX3-CDS (n = 5), and both faster, although not significantly (P = .06), than MA9 mice (n = 5). (F) Quantitative PCR analysis of miR-181b and PBX3 expression levels in secondary BMT mouse leukemic BM cells. The quantitative PCR primers of PBX3 were designed to detect expression of both human and mouse PBX3. Data are mean ± SD values of 3 mouse BM samples per cohort.