Knockdown of Stat5 reduces proliferation and alters cell cycle of leukemic cells. (A) Cells (606HS2 and 931HS2) were infected with a lentiviral vector transducing Stat5-shRNA17 or Shp2-shRNA19 or control ns-shRNA and sorted for green fluorescent protein expression. Experiments were performed 72 hours after infection. Whole-cell extracts were immunoblotted with anti–P-Stat5 and anti-Stat5 Abs and anti–β-actin Ab as a loading control. (B) Cells were plated at 1 × 105 cells/mL. Viable cells were evaluated by trypan blue exclusion at 24 and 48 hours. Data are means ± SEM (n = 4, performed in duplicate). (C) Representative colonies grown into methylcellulose for 6 days. Bars correspond to 80 μm. The average number of colonies per 100 seeded cells ± SEM is indicated below (n = 3, performed in duplicate). (D) Subcutaneous tumors in nude mice engrafted with 606HS2 and 931HS2 cells transducing Stat5-shRNA17 or control ns-shRNA were taken 3 weeks after injection and weighed. Bar represents the mean weight ± SEM of 8 tumors. (E) Representative cell-cycle distribution of green fluorescent protein–sorted cells plated at 1 × 105 cells/mL for 24 hours. The percentages of living cells in the G0/G1, S, and G2/M phases of the cell cycle are indicated below. The values are from 1 of 3 independent experiments performed at 48 hours. Similar results were obtained when experiments were performed at 24 hours (not shown).
