includes tumor stem cells [12,13,17]. Later studies have shown that additional ABC transporters are involved in drug resistance of SP cells. ABCA3 is highly expressed in AML patient samples and its expression is associated with unfavorable clinical treatment outcome [23]. Furthermore, the expression of ABCA3 is enriched in leukemic SP cells and has been linked to multidrug resistance by facilitating lysosomal sequestration of drugs in AML primary cells and cell lines [23,24,14,15,20]. Previously, we have shown that SALL4, a zinc finger transcription factor, forms a core transcriptional network with Oct4, Nanog and Sox2, which governs the self-renewal property of murine embryonic stem (ES) cells [25,26]. In addition, we found that during normal hematopoiesis, SALL4 is preferentially expressed in human CD34+ hematopoietic stem/progenitors (HS/PCs) and down-regulated in CD34- cells during hematopoi- etic differentiation [27]. Furthermore, SALL4 is aberrantly expressed in human acute myeloid leukemia, and transgenic SALL4 mice develop acute myeloid leukemia [27]. Loss-of- function studies have demonstrated that SALL4 is a key regulator in leukemic cell survival and down-regulation of SALL4 led to significant apoptosis of leukemic cells [28]. The important role of SALL4 in normal HSC and leukemic stem or initiating cells is supported by its interactions with several key players in self- renewal of HSCs and LSCs: Wnt/b-catenin [27], Bmi-1 [28,29], and PTEN [30]. In this study, we reported that SALL4 expression was higher in drug resistant primary acute myeloid leukemic patients than those from drug-responsive cases. In addition, while overexpression of SALL4 led to drug resistance in cell lines, cells with decreased SALL4 expression were more sensitive to drug treatments than the parental cells. Having observed that SALL4 was enriched in HS/ PCs that were identified by cell surface markers, we planned to evaluate the expression of this stem cell factor in SP and non-SP cells in various blood cancer cell lines (HL-60, NB4, KG1a and RPMI8226 cells), as well as investigate the role of SALL4 in drug resistance and cancer stem cells as identified by SP studies. We found that SALL4 expression was enriched in the SP when compared to the non-SP counterpart. We also found that SALL4 could promote the expression of the ABC transporter genes, such as ABCA3 and ABCG2, and knocking down of SALL4 expression led to a decreased SP. These observations suggest that SALL4 can contribute to the SP phenotype by regulating the expression of ABCA3 and ABCG2. Therefore, by targeting SALL4, we may be able to target the leukemic SP or LSC population. Materials and Methods Flow cytometry analyses For side population studies, the cells (16106/ml) were incubated at 37uC for 90 min with 5 mg/ml Hoechst 33342 (Invitrogen), either alone or in the presence of 50 mM verapamil (Sigma) as a negative control. Side population cells were analyzed on a flow cytometer equipped with 424/444 nm band pass, 670 nm long pass and optical filter. Dead cells were excluded by gating on forward and side scatter which eliminated the PI-positive population. The data were analyzed by FlowJo (Ashland, OR, USA). Freshly sorted SP cells were counted, plated in six-well plates, and cultured with RPMI1640 for 7–8 days. Expanded SP cells were transduced with retrovirus for knocking down of SALL4 and reanalyzed for side population as described below. Cell lines and cell culture All cell cultures were maintained at 37uC with 5% CO2. Several human malignant hematopoietic cell lines, NB4, HL-60, KG1a, SALL4 Affects the Side Population RPMI8226, 293(embroyonic kidney), MCF7 (breast cancer) and HeLa cell lines were obtained from American Type Culture Collection Inc (ATCC, Lawrenceville, GA, USA). Human CD34+ cells CD34+ cells were purified from primary human blood samples obtained from Lonza Walkersville Inc (Maryland, USA). Briefly, the samples were spun down at 2,400 rpm for 5 seconds at 4uC and the supernatant was discarded, the pellet was then resuspended in 1 mL of 16PBS supplemented with protease inhibitor cocktail. The cells were then sorted using the EasySep Human Whole Blood/Buffy Coat CD34 Positive Selection Kit (Stemcell Technologies, Vancouver, Canada). 200 mL of the EasySep CD34 antibody serum was added to the sample and incubated at room temperature for 15 minutes, then 100 mL of the EasySep magnetic bead mixture was added and incubated at room temperature for 10 minutes. 3 mL of the sample was selected in a 12675 mm round-bottom polystyrene tube (BD Falcon, San Jose, CA) using ‘‘The Big Easy’’ EasySep Magnet (Stemcell Technol- ogies). Selection occurred when the sample in the tube was incubated in the magnet at room temperature for 5 minutes, and then the supernatant was discarded. The remaining sample was washed three times with 1.5 mL of 16PBS supplemented with 2% FBS and 1 mM EDTA, each with a 5 minute incubation period in the magnet between discarding of the supernatant. ChIP (Chromatin immunoprecipitation) 293 cells were transiently transfected either with a full length SALL4A, SALL4B or an empty vector. Cells were cross-linked, lysed, and sonicated. Chromatin immunoprecipitations were conducted with a polyclonal anti-SALL4 antibody as described before [28,29], followed by quantitative real-time PCR. Three sets of ABCA3 and two sets of ABCG2 primers were used to validate the pulldown DNA fragments. Fold enrichment was calculated after normalization with input (no antibody added) of transfected cells. The ABCG2 primers for PCR were 59- CCTGGATGTCCGGGTCTC-39 (forward-1), 59-AACCTTTTGAGTGGGCACAG-39 (reverse-1), 59-CTGTG- GAGGAACTGGGTAGG-39 (forward-2), 5-CAAAGGCTCAG- GATCTCAGG-39 (reverse-2) and for ABCA3 was 59-TCTGGG- ACGAAGCAGAGAAT-39 (Positive forward), and 59- TGAGCA- TACAGGGGGAAAA-39 (Positive reverse). 59-TCTGAGTCAGC- CAGTTCTGGTA-39(Negative1-forward), 59-CACCCGGTCAT- ATCCTCAGT-39(Negative1-reverse), 59-TTTGGCATTTATGT- GTCAGGT-39(Negative2-forward), 59-TGAGTCTCAATTTCC- TATCCCTAAA-39(Negative2-reverse). Quantitative real-time PCR (qPCR) Pulldown DNA from ChIP was measured by UV spectropho- tometry. About 60 ng of DNA was utilized to perform qPCR with iTaq SYBR Green Supermix (Bio-Rad). Reactions were carried out in duplicates. Quantitative real-time RT-PCR (qRT-PCR) Total RNA was extracted with Trizol reagent (Invitrogen) or RNeasy Micro Kit (Qiagen) according to manufacturer’s instruc- tions, and the concentration was measured by UV spectrophotom- etry. 100 ng of total RNA was applied to conduct qRT-PCR by using iScript One-Step RT-PCR Kit with SYBR Green (Bio-Rad). The average threshold cycle for each gene was determined from duplicate reactions and the expression level was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). SALL4 primers (amplicon length, 68 bp) were the following: 59- TGCAG- CAGTTGGTGGAGAAC -39 (forward); 59-TCGGTGGCAAAT- PLoS ONE | www.plosone.org 2 April 2011 | Volume 6 | Issue 4 | e18372