The Role of GATA1s in DS AMkL Figure 1. Down-regulation of GATA1s in CMK cells results in impaired cell proliferation. Expression of GATA1s in two selected subclones, CMK-5a and -5b, in comparison to the negative control (CMK-neg) was verified by Western blotting (panel A) and real-time RT-PCR (panel B). The real-time RT-PCR results were expressed as mean values 6 standard errors from 3 independent experiments using the same cDNA preparation and normalized to GAPDH. To establish the doubling times for each shRNA subclone, the CMK-5a, -5b, and –neg sublines were seeded at 2.56104 cells/ml and counted every 24 h with trypan blue staining (panel C). Cell cycle progression in the CMK-5a, -5b, and –neg sublines was assessed by PI staining and flow cytometry analysis, as described in the Materials and Methods (panel D). DNA content was also assessed in the CMK-5a, -5b, and –neg sublines by incorporating BrdU into DNA and flow cytometry analysis as described in the Materials and Methods (panel E). doi:10.1371/journal.pone.0027486.g001 establish that GATA1s represses cell differentiation towards the megakaryocytic lineage in DS AMkL. Down-regulation of GATA1s in CMK Cells Increases Cytotoxicities to Ara-C, Daunorubicin, and VP-16 To determine the role of GATA1s in ara-C, daunorubicin, and VP-16 sensitivities, in vitro drug cytotoxicities were determined in the CMK shRNA stable clones with decreased levels of GATA1s. Sensitivities to ara-C for the CMK-5a and CMK-5b cells, as reflected in IC50 values, were 2.1- and 2.5- fold, respectively, greater than that for the CMK-neg cells (Fig. 3A). Essentially the same results were obtained for daunorubicin and VP-16 in the shRNA stable clones (data not shown). This suggests that expression of GATA1s confers resistance to standard chemother- apeutic drugs in DS AMkL rather than enhancing their sensitivities. Hence, loss of GATA1 rather than expression of GATA1s is likely an important factor accounting for the enhanced chemosensitivity in DS AMkL compared to non-DS AML, leading to high EFS rates.[16] To begin to determine the molecular mechanism(s) responsible for the changes in ara-C sensitivity accompanying decreased expression of GATA1s, we used real-time RT-PCR to quantify transcript levels for genes encoding ara-C metabolizing enzymes, dCK and CDA, and for hENT-1, which encodes the major membrane nucleoside transporter of ara-C. Interestingly, there were no consistent patterns for transcript levels for dCK and hENT- 1 accompanying decreased GATA1s (data not shown). CDA transcripts were not detectable in the CMK stable clones (data not PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27486