GAGACATTC -39 (reverse); and GAPDH primers (amplicon length, 120 bp): 59- TGTACGCCAACACAGTGCTG -39 (for- ward); 59- TCAGGAGGAGCAATGATCTTG -3 (reverse). ABCA3: 59-CTCCGAGAAGGACTTTGAGG-39 (forward); 59- TCCGTGTGTAACTGAACCGT-39 (reverse), and ABCG2; 59- CCCAGGCCTCTATAGCTCAG-39 (forward); 59-CGTCAG- GAAGAAGAGAACCC-39 (reverse). The expression levels of SALL4 in AML patients were analyzed by 2-DDCt relative quantitative method. Retrovirus production and SALL4 knock-down The specificity of the SALL4 shRNA has been described in our previous publications [28,31]. Briefly, three short-hairpin RNA- expressing plasmids, one control (GFP-pRS) and two SALL4 specific (no. 7410, no. 7412; all 3 from Origene, Rockville, MD), were transfected into Phoenix packaging cells (Orbigen, San Diego, CA) using Lipofectamine 2000 (Invitrogen, Frederick, MD). Virus was harvested 48 hours after transfection and filtered through a 0.45-mm filter. Virus supernatant was added to KG1a or CD34+ cells with 8 mg/ml polybrene. Cells were centrifuged at 2506 g for 2 hr and incubated for 90 min. After 90 min of incubation, virus supernatant was replaced with fresh medium and incubated for 24 hr. MTT assay and drug treatment 2000 cells were seeded into 96-well plates in RPMI 1640 containing 10% FBS. After 20–24 hr, cells were replenished with fresh complete medium containing either a test compound or H2O as a vehicle. At the indicated time, the cell proliferation reagent MTS-1 (Promega) was added to each well. The assay was measured at 450 nm using an ELISA Reader (Millipore). Patient Study Population Ethics Statement. Discarded peripheral blood samples were obtained from 68 AML patients (AML M2: 42; AML M3: 12; AML M4: 5, AML unclassified: 9) and 30 healthy individuals, between December 2007 and May 2008, in the Peking Union Medical College Hospital, under an approved protocol from the IRB of Peking Union Medical College Hospital. Informed consent was not obtained due to the discarded nature of these samples and the data were analyzed anonymously. The diagnosis was based on standard cytological criteria according to the French–American–British (FAB) classification [32]. Patient diagnosis was sub-classified by morphology and by immunophenotyping by two independent pathologists. Patient age at diagnosis ranged from 18 to 82 years with a median of 44 years. The AML patients were given standard chemotherapy with most of them responding to the treatments. Ages of the healthy individuals ranged from 14 to 65 years with a median of 41 years. The 461-AML-cohort was downloaded from Gene Expression Omnibus dataset GSE6891. The data was then analyzed using ParteK Genomic Suite (Missouri, MO, USA). The mean intensity of SALL4 was calculated for the dataset, and each sample was then stratified as SALL4 high or SALL4 low according to their SALL4 expression value. Statistical Analysis SPSS software (version 11.0, SPSS Inc.) was used for statistical analysis of SALL4 expression. All results were presented as a median value (quartile 1, quartile 3) because the SALL4 expression in leukemia showed a skewed distribution. Using the Mann-Whitney test, we compared the SALL4 expression between AML in different treatment stages and the healthy control, SALL4 Affects the Side Population between pre-treatment and post-treatment AML patients. The Pearson correlation coefficient between SALL4 expression and AML treatment and status was determined. In all cases, a P value ,0.05 was considered statistically significant. Statistic comparisons of the ABC genes in SALL4 high- or SALL4 low- group were made using student t-test. Electrophoretic Mobility Shift Assays (EMSAs) DNA-protein binding assays were carried out with nuclear extract from SALL4B transfected HeLa cells prepared with Nuclear Extract kit (Active Motif) according to manufacturer’s instructions. Synthetic complementary oligonucleotides were 39-biotinylated using the biotin 39-end DNA labeling kit (Pierce) according to the manufacturer’s instructions. The sequences of the oligonucleotides used are 59- GCAGAGAATGGGGAAGACATTTTGGATGG- GGTGTTGCAG-39 (#1), 59- TCTGTGAAGACTGATGGG- GAAAGGGGGTTTTTAGGATGTT-39 (#2) and 59-AAGTCA- GAGCGCCCTCTGCCGGGCATGGGCAGCATGCAGG-39 (#3) for ABCA3. Binding reactions were carried out for 20 min at room temperature in the presence of 50 ng/ml poly (dI-dC), 0.05% Nonidet P-40, 10 mm EDTA, and 2.5% glycerol in 16 binding buffer (LightShiftTM chemiluminescent EMSA kit, Pierce) using 20 fmol of biotin-end-labeled target DNA and 2 mg of nuclear extract. Unlabeled target DNA (4 pmol) or 2 ml of anti-SALL4 antibody [28] or anti-Tubulin antibody (Sigma) was added per 20 ml of binding reaction as indicated. Assays were loaded onto native 6% polyacrylamide gels pre-electrophoresed for 60 min in 0.56 Tris borate/EDTA and electrophoresed at 100 V before being trans- ferred onto a positively charged nylon membrane (HybondTM-N+) in 0.56Tris borate/EDTA at 100 V for 30 min. Transferred DNAs were cross-linked to the membrane at 120 mJ/cm2 and detected using horseradish peroxidase-conjugated streptavidin (LightShiftTM chemiluminescent EMSA kit) according to the manufacturer’s instructions. Additional method is listed Text S1. Results SALL4 expression is associated with AML treatment status We have shown that SALL4 is aberrantly expressed in AML patients [33,27]. In this study, we sought to investigate whether SALL4 expression level is associated with AML treatment status. Using the optimized qRT-PCR condition that we established earlier [33,27,28], we compared SALL4 expression level in AML patients without treatment (N = 36), AML patients in the complete remission (CR) phase (N = 25), AML patients in the partial remission (PR) phase (N = 7), and healthy controls (N = 30). The definition of CR or PR is based on blast count after the completion of standard chemotherapy. The blast count in bone marrow of the patients in AML CR phase was #5%, the blast count in bone marrow with the patient in AML PR phase was between .5% and #20%. We noticed that SALL4 had the highest expression level in acute AML patients without treatment [69.01 (range: 17.20,120.28)], low expression level in PR patients [4.18 (range: 1.76,5.04)], even lower expression level in CR patients [1.82 (range: 0.50,2.96)], and the lowest expression level in healthy controls [0. 80 (range: 0.35,1.13)]. The median level of SALL4 mRNA expression in patients with acute AML without treatment was 86-fold higher than that in the healthy controls (Z = 26.89, p = 0.001), 38-fold higher than that in the AML CR phase (Z = 26.34, p = 0.001), and 17-fold higher than that in the PR phase (Z = 23.61, p = 0.001). The level of SALL4 expression in the AML PR phase was 2-fold higher than that in the AML CR phase (Z = 22.44, p = 0.015, Figure 1A). PLoS ONE | www.plosone.org 3 April 2011 | Volume 6 | Issue 4 | e18372