compared to untreated control cells. The data are presented as mean values 6 standard errors from a minimum of 3 independent experiments. Assessment of Baseline Apoptosis The CMK shRNA stable clones were harvested, vigorously pipetted, and triplicate samples were used for determining baseline apoptosis with the Apoptosis Annexin-V FITC/PI Kit (Beckman Coulter; Brea, CA), as previously described.[18,19] Apoptotic events were recorded as a combination of Annexin-V+/PI- (early apoptotic) and Annexin-V+/PI+ (late apoptotic/dead) events. Western Blot Analysis Soluble protein extracts were prepared by sonication in hypotonic buffer (10 mM Tris-Cl, pH 7.0), containing 1% SDS and proteolytic inhibitors, and subjected to SDS-PAGE. Separated proteins were electrophoretically transferred to PVDF membranes (Thermo Fisher Inc., Rockford, IL) and immunoblotted with anti- GATA1, -Bax, -Bad, -Bid, -Bcl-xL, –Bcl-2 (Cell Signaling Technology, Danvers, MA), or -b-actin (Sigma-Aldrich, St. Louis, MO) antibodies, as described previously.[16] Immunoreactive proteins were visualized using the Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE), as described by the manufacturer. Gene Expression Microarray Analysis Gene expression microarray was performed with the Agilent Whole Human Genome 4x44K microarray, catalog #G4112F. Microarray sample preparation, hybridization, and data analysis were described previously.[20] On each microarray, a labeled CMK-5a or -5b sample was co-hybridized with an oppositely labeled CMK-neg sample. Two arrays were completed for the CMK-5a/CMK-neg pair and the CMK-5b/CMK-neg pair, respectively, for a total of four arrays. The two microarrays used for each clone were hybridized in a dye swap arrangement with opposite dye orientation to minimize the dye bias effect. Statistical analyses were performed using Rosetta ResolverH.[21] We have deposited the raw data at GEO under accession number GSE32388, we confirm all details are MIAME compliant. To identify overlapping probes/genes between the CMK gene set and our previously reported gene set derived from a comparison between DS and non-DS AMkL primary patient samples,[22] the probe IDs of each gene set were cross referenced to Entrez Gene IDs, and a common set of Entrex IDs between the 2 groups was identified. Quantification of Gene Expression by Real-time RT-PCR Transcripts for GATA1, Bcl-2, deoxycytidine kinase (dCK), cytidine deaminase (CDA), human equilibrative nucleoside transporter 1 (hENT1), interleukin 1 alpha (IL1A), platelet factor 4 (PF4), and tubulin, beta 1(TUBB1) were quantitated using a LightCycler real-time PCR machine (Roche, Indianaopolis, IN), as previously described,[16] or using Taqman probes (Applied Biosystems Inc, Foster City, CA, for PF4 and TUBB1). Primers and PCR conditions are described in Table S1. Real-time PCR results were expressed as mean values 6 standard errors from 3 independent experiments using the same cDNA preparation and normalized to GAPDH (glyceraldehyde 3- phosphate dehydrogenase) transcripts. Chromatin Immunoprecipitation Assays The chromatin immunoprecipitation (ChIP) assay was per- formed as described previously.[23,24] Purified chromatin frag- ments from CMK cells were incubated with anti-GATA1 antibody or normal IgG. Standard PCR (qualitative) for the GATA1 The Role of GATA1s in DS AMkL binding region in the IL1A promoter was performed with forward (59-GGGGAATTATTTACAACAGAGGAGTG-39) and reverse (59-TAATGCCTCAGTTCACCAAAGAAA-39) primers span- ning the three putative GATA1 binding sites. An upstream unrelated (ie, to GATA1 binding) region in the IL1A gene was amplified by PCR with the use of upper (59-CTACATCAAT- CACCCATCTCCA-39) and lower (59-CTTCACGTTCAGT- CAGCAAAT-39) primers to further validate the specificity of the ChIP assays. The same IL1A promoter regions were also amplified by real-time PCR (quantitative), using the same primers, on a Roche LightCycler-480 real-time PCR machine. Effects of Recombinant IL1a on Cell Proliferation of CMK- 5a Cells CMK-5a cells were seeded at a density of 2.56104 cells/mL in T25 flasks and either recombinant IL1a (ProSpec, Rehovot, Israel) reconstituted in PBS, or an equal volume of PBS was added. The cells were cultured for up to 48 h at 37uC and counted in triplicate every 24 h. Statistical Analysis Student’s t test (two tail, unequal variance) was used for statistical analysis on experiments unless otherwise specified. Results GATA1s Promotes Cell Proliferation and Represses Megakaryocytic Differentiation of CMK Cells To investigate the role of GATA1s in DS AMkL, we used lentiviral shRNA to knockdown the GATA1 gene in a DS AMkL cell line, CMK, established from a 1-year-old DS boy with AMkL and harboring a mutated GATA1 gene.[17] shRNA lentiviral infection resulted in 59% and 30% decreased levels of GATA1s in two clones, designated CMK-5a and -5b, respectively, compared to a pool of cells from the negative control infection (designated CMK-neg, Fig. 1A). This was accompanied by parallel changes in transcript levels for GATA1 in the CMK-5a and -5b cells, compared to the CMK-neg cells (Fig. 1B). Down-regulation of GATA1s was accompanied by GATA1s dose-dependent de- creased proliferation of the CMK-5a and -5b stable clones (doubling times 36.4 and 28.2 h, respectively), compared to the CMK-neg cells (25.8 h, Fig. 1C). Interestingly, this was unlikely related to cell cycle perturbations since only minimal changes of the cell cycle phases (e.g., slight decreases in number of cells in S phase) in the CMK-5a and -5b clones were detected by PI staining and flow cytometry analyses, compared to the CMK-neg cells (Fig. 1D). The lack of obvious changes in S phase in the CMK GATA1 shRNA stable clones was further confirmed by BrdU incorporation assays (Fig. 1E). These results demonstrate that GATA1s promotes cell proliferation in DS AMkL. To determine the role of GATA1s in megakaryocytic differentiation, flow cytometry analysis of megakaryocytic-associ- ated antigens (e.g., CD61 and CD41) on the cell surface was performed in the CMK-5a, -5b, and –neg cells. Interestingly, down-regulation of GATA1s in CMK cells resulted in a significantly increased total cell surface expression of CD61 (isotype ratio of 36.9 and 13.5, respectively) and CD41 (isotype ratio of 14 and 6.6, respectively) as a measure of megakaryocyte maturation in the CMK-5a and CMK-5b clones relative to the CMK-neg cells (isotype ratio of 7.8 and 3.3, respectively; p,0.01) (Fig. 2). Total cell surface expression of glycophorin-A was significantly decreased in the CMK-5a and CMK-5b clones (isotype ratio of 2.1 and 1.8, respectively), compared to the CMK- neg cells (isotype ratio of 17.5; p,0.01) (Fig. 2). These results PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27486