Knockdown of L3MBTL1 promotes the erythroid differentiation of human hematopoietic CD34⁺ progenitor cells. (A) Lentiviral constructs expressing shRNAs targeting luciferase (control) or L3MBTL1 (sh1 and sh2) led to efficient knockdown in primary CB CD34⁺ cells, as assessed by Western blot and quantitative RT-PCR. shRNAs of a different backbone (empty vector and sh3) also yielded efficient knockdown of L3MBTL1. All the presented data were confirmed using both sets of constructs. (B) Expression of CD71 and GlyA on human HSPCs, as assayed by flow cytometric analysis at days 7, 9, and 11 of Epo-induced culture. (C) Expression of CD71 and GlyA on human HSPCs, cultured with different concentrations of Epo, as assayed by flow cytometric analysis at 7 days. (D) Cells from panel B were stained with May-Grunwald-Giemsa on day 7 of Epo-induced culture, and their morphology was captured by light microscopy. (E) Epo-exposed cells at days 7, 9, and 11 of Epo-induced culture were resuspended in benzidine solution, and cells that stained dark blue-green were scored as positive.