STAT5-induced long-term proliferation and increased CFC and LTC-IC frequencies are impaired by down-modulation of HIF2α. (A) Cord blood CD34⁺ cells were transduced with MiNR1 control or STAT5A-ER together with luciferase RNAi control or HIF2αRNAi constructs, and double-transduced cells were sorted. Sorted cells were stimulated with 100nM 4-OH for 24 hours, and then RNA was isolated. HIF2α mRNA expression levels were measured by Q-PCR. (B) Cord blood CD 34⁺ cells were transduced and sorted as described in panel A, but were used for Western blot analysis to determine HIF2α protein levels. (C) Double-transduced cord blood CD34⁺ cells as described in panel A were cultured in MS5 coculture. Cocultures were demi-depopulated weekly and cumulative cell counts are indicated. (D) Suspension cells from cocultures as described in panel C were analyzed in CFC assays. Data indicate total CFC numbers from a representative experiment of 3 independent experiments. (E) LTC-IC frequencies were determined in limiting dilution on MS5 stromal cells. Cultures were cultured for 5 weeks, and then methylcellulose was added. At week 7, LTC-IC frequencies were determined. Data shows the stem-cell frequencies of a representative experiment of 3 independent experiments. (F) RNA was isolated from double-transduced cells as described in panel A, RNA was isolated, and real-time Q-PCR was performed to identify HIF2α target genes. CFU-GM, colony-forming unit–granulocyte-macrophage; BFU-E, burst-forming unit–erythroid; *P < .05.