Efficient STAT5 knockdown in CD34⁺ AML cells impairs long-term growth on MS5 coculture. (A) Western blot analysis for phospho-STAT5 in 8 AML patients. Actin is shown as loading control. Flt3-ITD mutations are indicated, as well as the fold down-regulation of expansion after STAT5 RNAi. (B) Transduction efficiency of CD34⁺ AML cells, performed as indicated in “Lentiviral transductions.” (C) Western blot analysis of STAT5 levels in control (Renilla) or STAT5 RNAi–transduced CD34⁺ cells. Percentages indicate expression of STAT5 relative to control cells, normalized against STAT3 or actin levels. Quantitative Western blot analysis was performed on an Odyssey infrared scanner or using Quantity One imaging software from Bio-Rad. (D) Q-PCR analysis of the expression of the STAT5 target genes CIS, SOCS 1, and SOCS 2 after STAT5 RNAi in transduced TF1 and AML CD34⁺ cells. (E) Cumulative cell counts of control (Renilla) or STAT5 RNAi–transduced CD34⁺ AML cells on MS5 coculture. All cell counts were normalized against week 0, the time of plating. Four representative cultures are shown. N = 11. (F) Transduced CD34⁺ AML cells in long-term coculture on MS5 stromal cells. Shown are average YFP (Renilla control) or GFP STAT5 RNAi percentages relative to week 1. A trendline is indicated and error bars denote standard deviations. N = 7. (G) Averaged calculated expansion of 7 AML MS5 cocultures, with error bars indicating standard deviations. The average expansion at week 6 is indicated next to the experimental group.