SALL4 Affects the Side Population Figure 2. Effects of SALL4 on drug sensitivity. Overexpression of SALL4 resulted in more resistance to doxorubicin (A) and daunorubicin (B) compared to control cells. 293 cells overexpressing SALL4B (filled circles) or an empty vector (open circles) were seeded in a 96-well plate and cultured with various concentrations of doxorubicin (A) or daunorubicin (B). At 48 hr after treatment, proliferation was measured with the MTS reagent (Promega). (C) Retroviral-mediated SALL4-knockdown KG1a or control cells were treated with doxorubicin. At 24 hr after drug treatment, cell proliferation was measured. Data is represented as mean 6 S.D. from three independent experiments. X axis: relative proliferation rate. Y axis: concentrations of doxorubicin or daunorubicin. doi:10.1371/journal.pone.0018372.g002 cells of each cell line by qRT-PCR. Expression of SALL4 was 4.2-, 3.0-, 2.2- and 3.0-fold higher in HL-60, RPMI8226, NB-4 and KG1a SP cells than those from the corresponding non-SP cells, respectively (Figure 3B). Furthermore, SALL4 protein expression was investigated in SP and Non-SP cells by an immunofluorescent stain in KG1a cell line. Consistent with the RNA results, SALL4 was more abundantly expressed in SP cells compared to non-SP KG1a cells (Figure S1). These data suggest that SALL4 is more abundant in SP cells compared to non-SP cells. Knocking-down of SALL4 leads to reduced frequency of the side population To determine if SALL4 is necessary for SP cell maintenance, we next performed loss-of-function study of SALL4 in side population. We first sorted SP cells from KG1a cells. Due to the low number of SP cells, we cultured the sorted SP cells with media containing 10% FBS and expanded them for a week. The SP cells could give rise to both SP and non-SP cells during this culture system, a character that is only observed in SP cells. On the other hand, culture of the non-SP cells yielded no non-SP cells (Figure S2). In addition, in vivo transplantation experiments showed that the KG1a SP population had an increased leukemic engraftment when compared to that of the non-SP cells in a xenotransplant mouse model. Four months after injection of 16105 of SP or non-SP cells into sub-lethally irradiated NOD- SCID mice, the SP recipients had 37% human CD45 cells detected in their bone marrows that were derived from the SP cells, while the non-SP recipients had only 0.1% human CD45 cells (Figure S3). In summary, these features suggest that the SP cells that we sorted from the KG1a cell line are enriched for leukemic stem cell properties and are consistent with other reports. The expanded SP cells were then infected with retroviruses containing shRNA against SALL4 or control scramble shRNA using the same approach as described previously [28]. The SP cell quantification was re-analyzed. qRT-PCR analysis showed that SALL4 expression was reduced by 40%. SALL4 knock-down significantly decreased the percent- age of the SP cells from 1.31%to 0.36%. These data suggest that SALL4 plays an important role in the maintenance of side population (Figure S4). PLoS ONE | www.plosone.org 5 April 2011 | Volume 6 | Issue 4 | e18372