The Role of GATA1s in DS AMkL shown). Similar results were obtained for the protein levels for dCK and hENT1 (Fig. 3B). These results clearly demonstrate that the effects of GATA1s on ara-C sensitivity in DS AMkL must involve mechanism(s) unrelated to altered expression of major genes involved in ara-C transport and metabolism. The lack of changes in expression of hENT1, CDA and dCK among the CMK-5a, -5b, and –neg stable clones led to a hypothesis that GATA1s can modulate ara-C sensitivity in CMK cells by regulating expression of genes related to apoptosis. Further, the increased sensitivities to daunorubicin and VP-16 of the CMK-5a and -5b stable clones compared to the CMK-neg cells also support these notions. To test this possibility, baseline apoptosis in the CMK-5a, -5b, and –neg clones was determined by Annexin-V FITC/PI staining and flow cytometry analysis. Basal apoptosis in both CMK-5a and - 5b cells was significantly higher (2.3-fold and 1.5-fold, respectively, p,0.05) than that in the CMK-neg cells and was inversely proportional to levels of GATA1s (Fig. 3C). These results strongly suggest that downregulation of GATA1s lowers the apoptotic threshold contributing to the increased ara-C sensitivity in CMK cells. Since chemotherapy drugs induce apoptosis in cancer cells primarily through the intrinsic apoptotic pathway, we per- formed Western blotting to identify changes in apoptotic factors potentially involved in the increased ara-C sensitivity associated with down-regulation of GATA1s. Interestingly, substantially decreased levels of the anti-apoptotic Bcl-2 protein were detected in the CMK-5a and -5b stable clones (decreases of 42% and 22%, respectively, relative to CMK-neg), while protein levels for other Bcl-2 family members, including Bcl-xL, Bad, Bax and Bid, were not altered (Fig. 4A). Surprisingly, transcript levels for Bcl-2 were increased approximately 2-fold in the CMK-5a and -5b clones compared to the CMK-neg cells despite decreased levels of the protein (data not shown). These results suggest that the net impact of GATA1s on Bcl-2 is post- transcriptional, although the mechanism responsible for this effect is unclear. To determine if Bcl-2 was the causal factor for the enhanced ara-C sensitivities in the CMK-5a and -5b clones compared to the CMK-neg cells, shRNA knockdown of Bcl-2 gene was performed in CMK cells (Fig. 4B). As expected, decreased expression of Bcl-2 in CMK cells resulted in significantly increased basal apoptosis Figure 4. GATA1s exerts its effects on ara-C sensitivity through modulating Bcl-2 protein levels in CMK cells. Panel A: Soluble proteins from the CMK-5a, -5b, and –neg sublines were subjected to western blotting and probed by anti-Bcl-2, -Bcl-xL, -Bax, -Bad, -Bid, or b-actin antibodies. Panel B: Expression of Bcl-2 in two selected subclones, CMK-b7 and –b8, in comparison to the negative control (CMK-neg) was verified by Western blotting. Panel C: Basal apoptosis in the CMK-b7, -b8, and –neg cells was determined by annexin V/PI staining and flow cytometry analysis, as described in the Materials and Methods. Panel D: The CMK-b7, -b8, and -Neg cells were cultured in complete medium with dialyzed fetal bovine serum in 96-well plates at a density of 86104 cells/ml, with a range of concentrations of ara-C at 37uC, and viable cell numbers were determined using the Cell Titer-blue reagent and a fluorescent microplate reader. The IC50 values were calculated as the concentrations of drug necessary to inhibit 50% growth compared to control cells cultured in the absence of drugs. The data are presented as mean values 6 standard errors from at least 3 independent experiments. * and ** indicate p,0.05 and 0.005, respectively. doi:10.1371/journal.pone.0027486.g004 PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27486