Knockdown of Shp2 reduces proliferation and induces apoptosis of leukemic cells. Cells (606HS2 and 931HS2) were infected with a lentiviral vector transducing Shp2-shRNA77 or Shp2-shRNA78 or a control ns-shRNA and selected for puromycin resistance. Experiments were performed 72 hours after infection. (A) Whole-cell extracts were immunoblotted with the indicated Abs and anti–β-actin Ab as a loading control. (B) Cells were plated at 1 × 105 cells/mL. Viable cells were scored by trypan blue exclusion at 24 and 48 hours. Data are means ± SEM (n = 3, performed in duplicate). (C) Representative colonies grown into methylcellulose for 6 days. Scale bars indicate 80 μm. The average number of colonies per 100 cells seeded ± SEM is indicated (n = 3, performed in duplicate). (D) Subcutaneous tumors in nude mice engrafted with 606HS2 and 931HS2 cells transduced with Shp2-shRNA78 or control ns-shRNA were taken 3 weeks after injection and weighed. Bar represents the mean weight ± SEM of 8 tumors for Shp2-shRNA78-606HS2 cells, ns-shRNA-606HS2 cells, and ns-shRNA-931HS2 cells. For Shp2-shRNA78-931HS2 cells, only 2 tumors were obtained. (E) Cells were plated at 1 × 105 cells/mL and the percentage of apoptotic cells was evaluated 48 hours later by flow cytometry using anti-active caspase 3 Abs. Bars represent means ± SEM (n = 3). Statistical differences from the value of the control (ns) are indicated as follows: *P < .05; **P < .01 (by Student t test).
