Lycat siRNA reduced hematopoietic and endothelial gene expression during ES cell differentiation into EBs. (A) Lycat mRNA knockdown was detected in day 4 EBs from siRNA ES cell clones by semiquantitative RT-PCR. VCi, retroviral vector control ES cell clone; 680-9 and 680-19, 2 independent Lycat siRNA ES cell clones derived from V2MM_232680 (Open Biosystems); 078-15 and 078-16, 2 independent Lycat siRNA ES cell clones from V2MM_103078 (Open Biosystems). (B) The Lycat siRNA knockdown in panel A was confirmed by Q-PCR. (C) The Lycat siRNA reduced expression of endothelial (Flk1 and CD31) and hematopoietic (Gata1, Gata2, Runx1, and Scl) genes using Q-PCR. (D) The Lycat siRNA reduced the formation of Flk1⁺, C-Kit⁺, C-Kit⁺ and CD41⁺, and CD31⁺ cells in day 4 EBs from all 4 siRNA ES cell clones by flow cytometry. These were calculated from 3 independent experiments except those of CD31⁺, which was calculated from 2 independent experiments. (B,C) Error bars represent standard deviations. (D-G) Numbers within quadrants indicate percentages of positive cells.