Knock down of ETO2 in L8057 cells. (A) Diagram of lentiviral construct used to express Eto2-directed shRNA (shETO2). cPPT indicates central polypurine tract; CTS, central termination sequence; and H1, H1 promoter. EF1α promoter and the eGFP reporter gene are shown. (B) Knock down of ETO2 protein expression (top panels) was assessed in L8057 cells infected with either control shRNA (Control, lefthand lane) or shRNA directed against ETO2 (shETO2, righthand lane) by Western blot analysis in 3 independent experiments. The blots were probed with anti-POL II antibody (bottom panels) to control for protein loading. Percentage knock down of normalized ETO2 protein expression is shown. (C,D) L8057 cells expressing a control shRNA or ETO2 shRNA were induced to undergo megakaryocytic differentiation with TPA. Cells were stained for acetylcholine esterase (AchE) activity after 4 days of induction (C). Images were acquired using an Olympus BX60 microscope with a QImaging camera (Surrey, BC) using an Olympus lens at 10×/0.5 and 40×/0.5 numeric aperture objectives. Openlab version 3 software (Improvision, Coventry, United Kingdom) was used for image acquisition, and images were exported into Adobe Photoshop version CS2 (Adobe Systems, San Jose, CA) for processing. Note the increased number of brown staining (AchE-positive) cells that are larger in size, indicative of greater megakaryocyte maturation, when cells are infected with the ETO2 shRNA. The percentage of AchE-positive cells counted in 3 independent experiments (D). Cells treated with DMSO served as a control for TPA induction. (E) Pf4 (left) and GPIIb (right) mRNA levels were quantitated relative to GAPDH levels by Taqman real-time RT-PCR in L8057 cells infected with virus expressing either control shRNA (▭) or shETO2 (). Data shown in panels D and E are the average of 3 independent experiments, and error bars represent 2 SD.