Distribution of mouse Lycat mRNA in tissues and cell populations detected by RT-PCR. (A) Lycat mRNA had highest expression in the AGM and heart among multiple mouse tissues including E12.5 AGM, E16 fetal liver (FL), adult liver (AL), spleen (Sp), bone marrow (BM), kidney (Kid), lung (Lu), pancreas (Pan), intestine (Int), muscle (Mus), heart (Hrt), and brain (Br). (B) Lycat mRNA was enriched in the Lin⁻Sca-1⁺C-Kit⁺ HSCs in BM. Cells were sorted by flow cytometry with lineage markers, Sca-1, and C-Kit for hematopoietic stem cells. Lineage⁻Sca-1⁻C-Kit⁻, L⁻S⁻K⁻; Lineage⁻Sca-1⁺C-Kit⁺, L⁻S⁺K⁺ (Figure S2A). (C) Lycat mRNA expression was higher in B cells. Cells were sorted by flow cytometry with mature hematopoietic lineage markers including T4 and T8a for T lymphocytes (T), B220 for B lymphocytes (B), TER119 for erythrocytes (Ery), and Gr-1 for granulocytes (Gr). (D) Lycat mRNA was enriched in the CD31⁺CD45⁻ endothelial cells in adult mouse BM. Cells were sorted from BM by flow cytometry with anti-CD31 and anti-CD45. (E) Lycat mRNA could be detected in embryoid bodies from day 0 to day 7 (D0 to D7). The morphology of EBs is presented in Figure S3. (F) Lycat mRNA was 2-fold more enriched in the Flk1⁺ cells in embryoid bodies from day 4 than those from day 5. Flk1⁺ cells were sorted from staged embryoid bodies by anti-Flk1. (G) Lycat mRNA was enriched in the Flk1⁺ cell population in embryoid bodies. Lycat mRNA could be detected in Flk1⁺hCD4⁺(Scl⁺) (F⁺S⁺) and Flk1⁺hCD4⁻(Scl⁻) (F⁺S⁻) but not in the Flk1⁻hCD4⁺(Scl⁺) (F⁻S⁺) and Flk1⁻hCD4⁻(Scl⁻) (F⁻S⁺) cells, which were sorted by flow cytometry with anti-Flk1 and anti-hCD4 (Figure S2B). The RNA levels were determined and normalized by GAPDH using quantitative RT-PCR (A-F) and using semiquantitative RT-PCR by β-actin as an internal RNA control (G). Error bars in panels A-F represent standard deviations.