Combinatorial RNAi targeting BCR-ABL and SHP2. (A) Schematic representation of the lentiviral transgene plasmid encoding anti-SHP2 and anti-Bcr-Abl shRNAs. (B) Target mRNA levels were measured by real-time RT-PCR 4 days after lentiviral transduction of K562 cells and normalized in comparison to GAPDH expression. The shRNA encoded by the respective lentivirus is indicated on the top of each bar. (C) Immunoblot of K562 cells transduced with anti-BCR-ABL and an anti-SHP2 plus anti-BCR-ABL shRNA-encoding lentivirus. Cells transduced with control dcH1-GL4-SR (lane 2), dcH1-b3a2_1-SR (lane 3), and b3a2_237SHP2 (lane 4) were lysed 6 days after transduction. Lane 1 shows untransduced K562 cells. The top panel shows an immunoblot with anti-SHP2-specific antibody, and the bottom panel the same membrane reprobed with anti-ERK2 antibodies as loading control. (D) Effects of anti-b3a2, anti-SHP2, and anti-b3a2 plus SHP2 shRNAs on proliferation of K562 cells. Numbers of trypan blue-negative cells are shown after transduction with lentiviruses encoding single (dcH1-GL4-SR, dcH1-b3a2-SR, dcH1-237SHP2-SR) or combined (b3a2-237SHP2) shRNAs as indicated (results from 2 experiments). One milliliter with 10⁴/mL cells was plated, cultures were split and fed twice a week, and calculated cell numbers are indicated. Triangles depict transduction with control virus dcH1-GL4-SR, and circles show transduction with viruses encoding dcH1-b3a2_1-SR, dcH1-237SHP2-SR, and dcH1-b3a2-237SHP2, respectively. (E) Effects of lentivirus-mediated shRNAs on colony formation of CML CD34⁺ cells. The diagram shows the relative ratio of RFP⁺ colonies under low as compared to high cytokine stimulation (GM-CSF + IL-3) as in Figure 5.The specific RNAi target is indicated on top of each bar. No RFP⁺ colonies were found under low cytokine concentration.