Identification of cyclin-A1 as target candidate byin silicoexpression analysis and RT-PCR quantification. (A) Seven candidate target genes predicted in silico: Heatmap of normalized probe sets. Each row represents one probe set, and each column represents one sample dataset. Red represents increased levels of expression; and blue, decreased levels of expression. The darkest shade of red/blue represents average expression ± 3 SDs. (B-C) Model-based expression of probe set 205899_at representing cyclin-A1. (B) Expression in AML LSCs compared with HSCs/CD34⁺ BM mononuclear cells, PBMCs, and nonhematopoietic somatic tissues. The dashed line represents the cut-off value of 69.2 (mean plus 3 SDs of the HSC samples). *P < .001 (explorative). (C) Expression in AML LSCs and corresponding blasts (P = .297). (D) Cyclin-A1 expression quantified by quantitative RT-PCR. The dashed line represents the cut-off value of 0.048 (mean plus 3 SDs of the BM samples). (E) Immunofluorescence staining of cyclin-A1 in primary AML samples (representative example). Shown is a healthy BM (top row) and primary leukemic blasts (bottom row). Cells were stained with 4,6-diamidino-2-phenylindole (DAPI; left) and indirectly for cyclin-A1 (AlexaFluor-488, right). Negative controls without primary antibody and nonspecific isotype showed no fluorescence in both samples (not shown). Images were captured using a Nikon Eclipse E800 microscope. (F) Intracellular FACS staining of cyclin-A1 in primary AML blasts (representative example). BM mononuclear cells of a patient with cyclin-A1-expressing AML after gating out doublets in forward scatter (FSC)-area/height (A/H) and SSC-A/H plots. Top row: gating on blasts based on SSC and CD45 expression. Bottom row: gating on CD33-positive cells. Histograms show cyclin-A1 (black line) and isotype control (shaded) in the blast population.