Figure 3. Downregulation of SALL4 decreases HOXA9 expression in human primary AML and leads to apoptosis.

(A) Expression of SALL4 and HOXA9 was correlated in human primary AML samples. qRT-PCR was performed on 34 primary samples with primers for SALL4 and HOXA9 mRNA. Mean value of SALL4 was used to stratify the samples to SALL4^hi or SALL4^lo groups. Each dot represents a patient. Data are mean ± SD from 3 independent experiments. **P < 0.001. (B and C) Downregulation of SALL4 or HOXA9 expression in primary AML. Primary AML cells were transduced at a density of 1 × 10⁶ cells/ml with lentiviruses expressing scrambled, SALL4, or HOXA9 shRNA. After 2 days of culturing in media containing cytokines (10 ng/ml IL-3, 25 ng/ml SCF, and 10 ng/ml IL-6), transduced cells were selected with 1 μg/ml puromycin for 3 days on semisolid medium. Expression of SALL4 (B) and HOXA9 (C) in primary AML after SALL4, HOXA9, or scrambled shRNA treatment was measured by qRT-PCR. (D and E) Increased apoptosis after downregulation of SALL4 or HOXA9 using the approach described above in primary AML cells was further evaluated by TUNEL assay (D) and Trypan blue staining (E). Original magnification, ×20 (E). Data are mean ± SD from 3 independent experiments. *P < 0.05.