Blood, Vol. 110, Issue 10, 3601-3609, November 15, 2007

Mouse lysocardiolipin acyltransferase controls the development of hematopoietic and endothelial lineages during in vitro embryonic stem-cell differentiation
Blood Wang et al. 110: 3601

Supplemental materials for: Wang et al

Files in this Data Supplement:

• Table S1. PCR primers and product sizes (PDF, 49.7 KB)
  
  
• Table S2. Lycat-induced gene expression (PDF, 36.4 KB)
  
  
• Figure S1. Lycat is highly conserved in vertebrates (PDF, 27.2 KB) -
  The N-terminal amino acid sequences of zebrafish, fugu, human and mouse Lycat proteins are highly homologous, which the underlined (green) catalytic domains of H(X)₄D and EGTR are identical. The alignment was made using MultAlin ⁸⁸. DRLycat, zebrafish Lycat (BC066444); TNLycat, tetraodon nigroviridis Lycat (CAG10378.1); HSLycat, human Lycat (BX647953); MMLycat, mouse Lycat (XM_128781).
  
  
• Figure S2. Cell populations in bone marrow and EBs were sorted and used for determining Lycat mRNA expression by RT-PCR in Figure 1 (PDF, 303 KB) -
  (A) Different cell populations were sorted from the adult bone marrow by flow cytometry with lineage markers, anti-Sca1 and anti-C-Kit. R1, lineage-negative (Lin⁻ or L⁻); R1 and R2, Lin⁻Sca-1⁻C-Kit⁻ or L⁻S⁻C⁻; R1 and R3, Lin⁻Sca-1⁺C-Kit⁺ or L⁻S⁺K⁺. (B) Cell populations were sorted from D4 EBs derived from Scl:hCD4 R1 ES cells by FACS with anti-hCD4 and anti-Flk1. R4, Flk1⁺hCD4⁻/Scl⁻; R5, Flk1⁺hCD4⁺/Scl⁺; R6, Flk1⁻hCD4⁻/Scl⁺; R7, Flk1⁻hCD4⁻/Scl⁻.
  
  
• Figure S3. Morphology of embryoid bodies at day-0 to day-7 (PDF, 142 KB) -
  R1 ES cells were induced to differentiate into embryoid bodies as described in Materials and Methods.
  
  
• Figure S4. The Lycat transgene driven by the β-actin promoter, like the Flk1 promoter, also increased both hematopoietic and endothelial gene expression in EBs (PDF, 68.6 KB) -
  (A) Schematic representation of the -actin:Lycat which Lycat cDNA was driven by the β-actin promoter and a PGK-neo cassette was inserted (top); and the vector control that has no Lycat cDNA in the construct (bottom). (B) Overexprssion of Lycat increased hematopoietic (Gata1, Scl, Runx1) and hemangioblastic/endothelial (Bmp4, Flk1, CD31) gene expression in transgenic AC47 and AC48 ES clones using Q-PCR with SYBR Green. RNA levels were normalized by GAPDH.
  
  
• Figure S5. Neither overexpression of Lycat nor Lycat siRNA caused abnormal ES cell differentiation and apoptosis (PDF, 473 KB) -
  (A) Similar dynamic Flk1 expression was found in EBs from VC and FC5 ES cells at D2.75, D3, D3.25, D3.75 and D4. (B) No significant apoptosis difference was observed in D4 EBs from VC, FC2, FC5 and FC22 detected by flow cytometry analysis with Annexin V-FITC and PI. (C) Lycat siRNA did not lead to abnormal apoptosis in D4 EBs from all four siRNA ES cell clones analyzed by flow cytometry with Annexin V-FITC and PI. This represents one of two independent experiments.