Figure 2. SYK-directed shRNA induces differentiation in AML cell lines.

(A) Knockdown performance of shRNAs targeting SYK in HL-60 and U937 cell lines. Transcript levels were measured at 5 days post-infection using real-time PCR and are reported for each hairpin (Syk_1, Syk_7, and Syk_10) relative to the transcript level of a luciferase shRNA control (Luc). Error bars depict mean ±standard deviation (SD) across three replicates.

(B) HL-60 and U937 cells were infected with shRNAs targeting SYK and luciferase (Luc) and differentiation evaluated 7 days post-infection. A 32-gene differentiation signature was quantified by the LMA/bead-based approach and a Weighted Summed Score (Differentiation Score) calculated for all genes. Error bars denote mean ±SD of 16 replicates. The two SYK-specific shRNAs that induced the greatest degree of gene suppression also induced the highest Differentiation Score.

(C) May Grunwald Giemsa staining of HL-60 and U937 cells 10 and 12 days post-infection, respectively, with shRNAs targeting SYK demonstrates cellular differentiation when compared to a luciferase shRNA control. Images were acquired with an Olympus BX41 microscope, 1000X magnification under oil, and Qcapture software. The scale bar equals 25 μm.

(D) FACS analysis was performed with FITC and PE-labeled antibodies for CD11b and CD14, respectively. Six days post-infection with shRNAs targeting SYK, HL-60 cells were positive for single stained CD11b and CD14 and double staining compared to a luciferase control.