Shp2 expression inversely correlates with differentiation of hematopoietic cells. (A) Leukemic cells were treated with ATRA at 1 μM and collected at different time points to determine the percentage of cells in the S phase (top panel) and the Shp2 protein amounts by Western blot (bottom panel). (B) CD11b expression levels by FCM analysis. (C) In top panel, leukemic cells were treated with ATRA at 1 μM, and collected at 72 hours for analysis of Shp2 expression using double immunofluorescence labeling with anti-Shp2 (green) and Ki-67 (red) antibodies. Shown are leukemia cells before and after induction with ATRA. For morphologic analysis, cells untreated or treated with ATRA for 72 hours were collected and stained with Wright-Giemsa staining. The bottom panel shows NB4 cells treated with Shp2 AS (2 μM) for 0 and 72 hours. Images were viewed at 100 ×/0.3 NA with a Zeiss Axiovert S100 microscope. Images were captured with a SPOT 1.3.0 CCD camera and transferred to Adobe Photoshop 6.0. (D) NB4 cells were treated with Shp2 AS at 3 μM and collected at different time points for analysis of cell cycles with FCM. Error bars represent standard error. (E) CD11b expression levels on NB4 cells were determined by FCM analysis following treatment with Shp2 AS (2 μM) for 72 hours. The scale bar represents 10 μm. Data are representative of 3 independent experiments.