(A) qRT-PCR analysis of HoxA9 expression in SALL4B leukemic cells after HOXA9 shRNA or control scrambled shRNA (Scr-shRNA) virus infection following puromycin (1 μg/ml) selection for 72 hours on semisolid medium. (B) SALL4B leukemic cell colony formation and replating capacity after HoxA9 suppression. 1,000 HOXA9 shRNA– or scrambled shRNA–treated SALL4B leukemic cells were seeded on semisolid medium with puromycin (1 μg/ml) and replated every 10 days. The number of colonies per dish was recorded after each round of replating. (C) Total number of cells per dish after each round of replating. (D) Representative colony images 10 days after the first round of plating. Original magnification, ×10 (top); ×20 (bottom). (E and F) Increased cell death in HOXA9-knockdown cells was observed by TUNEL assay (E) and Trypan blue stain (F) 3 days after the first round of plating. Original magnification, ×20 (E). (G) Log-rank (Mantel-Cox) survival curve of mice transplanted with SALL4B leukemic cells treated with IF2-HOXA9 shRNA (n = 8), IF3-HOXA9 shRNA (n = 7), or scrambled shRNA (n = 11). Data are mean ± SD from 3 independent experiments. *P < 0.05, **P < 0.001.