SALL4 Affects the Side Population Figure 3. Relative expression of SALL4 in SP and non-SP cells from blood cancer cell lines. (A) Representation of FACS profile of SP cells from KG1a. A SP cell profile in the presence of verapamil (50 mM) was at the right as a control. (B) qRT-PCR analysis of SALL4 in SP (dark bars) and non- SP (open bars) cells from HL-60, RPMI8226, NB-4 and KG1a cells. For quantification of gene expression, amplification of GAPDH was performed as an endogenous control to standardize the amount of sample. Shown here are the representative results of at least two independent experiments. Y axis: relative SALL4 expression (fold) in comparison to non-SP. doi:10.1371/journal.pone.0018372.g003 SALL4 binds to the promoter region of ABCA3 but not ABCG2 gene in vivo It was reported that the ATP binding cassette (ABC) transporters can contribute to drug resistance. We have mapped SALL4 global gene targets using chromatin-immunoprecipitation followed by microarray hybridization (ChIP-on-chip) in myeloid leukemic NB4 cells [28], normal human CD34+ bone marrow cells, and 293 cells (unpublished data). ABCA3, but not ABCG2, was identified as a potential SALL4 downstream target gene in all three cell types. We then sought to validate whether SALL4 could bind to the promoter regions of ABCA3 but not ABCG2 quantitatively using regular ChIP coupled with qPCR. Based on the global chip array results with potential SALL4 binding sites, we designed 3 sets of primers for the ABCA3 promoter region (one was from a peak region, two sets were designed on non-peak regions for negative controls) and two sets of primers for the ABCG2 promoter regions (Figure 4A). 293 cells were transfected with a control vector or SALL4B construct. Total chromatin was isolated and fragmentized by sonication. Chromatin containing SALL4 protein was immuno- precipitated with a SALL4 antibody. To determine whether the promoters of ABCA3 or ABCG2 could be co-immunoprecipitated by the SALL4 complex, we performed PCR reactions to amplify the promoter regions. A fragment of the ABCA3 promoter region was detected in the SALL4 complex, in contrast, neither of the two ABCG2 promoter regions was detected (Figure 4A). Similar data was obtained using KG1a cells ((Figure S5). These data suggest that SALL4 can bind to the ABCA3 promoter directly but probably not to the ABCG2 promoter. To determine a specific protein-DNA interaction between SALL4 and the ABCA3 promoter, nuclear extracts from HeLa cells overexpressing SALL4B (Figure 4B) were used in EMSA assay. Based on our ChIP-qPCR result, we designed 3 probes PLoS ONE | www.plosone.org 6 April 2011 | Volume 6 | Issue 4 | e18372