Hematopoietic differentiation from HE is inhibited by depletion of SOX17. (A) Knock-down efficiencies of shRNAs against SOX17. Western blot analysis of SOX17 in 293T cells transduced with shRNAs against SOX17 (top panel). α-Tubulin was used as the loading control. pre-HPCs from day 8 EBs were transduced with shRNAs against SOX17 on OP9 cells and cultured in the presence of 20 ng/mL of SCF and TPO, 10 ng/mL of IL-3, and 3 units/mL of erythropoietin (EPO) for 7 days. Levels of endogenous SOX17 were analyzed by quantitative RT-PCR analysis (bottom panel). mRNA levels were normalized to GAPDH expression. Expression levels relative to that in the control cells transduced with an shRNA against Luciferase are shown as the means ± SD for triplicate analyses. (B) Effects of depletion of SOX17 on hematopoietic development from HE cells. ECs from day 5 EBs were transduced with shRNAs against SOX17 on OP9 cells and were cultured in the presence of 20 ng/mL of SCF and TPO, 10 ng/mL of IL-3, and 3 units/mL of EPO for 9 days. Representative flow cytometric profiles of cells at day 9 of culture are depicted. (C) Absolute numbers and proportion of CD235a⁺ erythroblasts and CD11b⁺ myeloid cells in panel B at day 9 of culture. Data are shown as the means ± SD for 3 independent cultures. (D) Effects of depletion of SOX17 on pre-HPCs. Pre-HPCs from day 8 EBs were transduced with shRNAs against SOX17 on OP9 cells and were cultured in the presence of 20 ng/mL of SCF and TPO, 10 ng/mL of IL-3, and 3 units/mL of EPO for 7 days. Absolute numbers and proportion of CD235a⁺ erythroblasts and CD11b⁺ myeloid cells at day 7 of culture are presented. Data are shown as the means ± SD for triplicate cultures of 1 of 2 independent experiments that gave similar results.