Human MA9 cells depend on Rac2 for growth and survival. (A) Western blot analysis of Rac2 knockdown efficiency using 3 shRNA sequences. Band densitometry is normalized to β-actin and NT control. (B) Cells were sorted for Venus⁺ cells on day 3 after transduction. Venus⁺ percentage was normalized to day 0 postsort. Plot is representative of 3 independent experiments using 2 different cell lines. (C) Lentiviral knockdown of Rac2 in MA9 cells induces apoptosis as shown by annexin V/7-AAD staining. Data are representative of 4 independent experiments. (D) Rac2 deficient MA9 cells exhibit decreased colony-forming ability in methylcellulose. Cells (10 000) were plated in triplicate and average colony count is shown from a representative of 4 separate experiments. (E) MA9 cells expressing Rac2 shRNA fail to expand in vivo. After lentiviral transduction, 12 500-20 000 FACS sorted Venus⁺ cells were injected intravenously into sublethally-irradiated NSS mice. Venus⁺ cell engraftment was measured by FACS analysis of BM aspirates at week 16. (F) Knockdown of Rac2 prevented leukemia deaths in a xenograft model. All NT control MA9 cell recipients died of Venus⁺ leukemia, whereas only 1 Venus⁺ MA9 leukemia death was seen in the knockdown groups. Two mice from the Rac2 knockdown groups died from Venus-negative MA9 AML, despite these cells representing < 2% of transplanted cells.