siRNA-mediated knockdown of OPN expression reduces cell survival in AML blasts and LSPCs. Purified CD34⁺ cells from the indicated AML patient samples were transfected with 50nM BLOCK-iT fluorescent oligo and 50 to 150nM of (1) RNAi High GC negative control duplexes; (2) scrambled siRNA duplexes; (3) OPN siRNAs duplexes no. 1, no. 2, or no. 3; or (4) a cocktail consisting of all 3 OPN siRNAs duplexes (A-B,D). After transfection, cells were cultured for 2 to 3 days in IMDM medium containing 0.5% FCS, after which time total RNA was isolated for quantitative RT-PCR (A) and viable cell number was assessed by using Flow-Count Fluorospheres (A,D). (B) Cell survival was determined by annexin V–Alexa 568 staining and flow cytometry. The survival of purified CD34⁺ cells isolated from patients with AML in culture varied from 15% to 90%, and results are presented as the percentage of cell survival relative to the control siRNA-transfected cells. The inset indicates the level of OPN mRNA expression for each AML sample as determined in Figures 4 and 7. (C) AML CD34⁺/CD38⁻/CD123⁺ LSPCs were purified by fluorescence-activated cell sorting (FACS). Cells were stained with CD34-FITC, CD38-PE-Cy7, and CD123-PE antibodies after which time LSPCs were sorted with FACSAria cell sorter into CD34⁺CD38⁻ subpopulation (1%-2% of total live cells) and gated for high CD123 expression (0.5%-1% of total). Approximately 5 × 10⁵ to 1 × 10⁶ cells were obtained from a single sort of 2 × 10⁸ thawed cells. The purity of sorted populations was confirmed by secondary flow cytometry. (D) LSPCs were transfected and analyzed for cell survival as described in panel A. †Samples were derived from relapsed patients.