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Blood, Vol. 109, Issue 11, 4777-4785, June 1, 2007
Blood and lymphatic endothelial cell-specific differentiation programs are stringently controlled by the tissue environment
Blood Amatschek et al.
109: 4777
Supplemental materials for: Amatschek et al, Vol 109, Issue 11, 4777-4785
Files in this Data Supplement:
- Figure S1. BEC-specific functional networks are lost in culture (PDF, 66.9 KB) -
Unbiased functional networks were generated by the Ingenuity software package based on gene and gene product interactions supported by experimental evidences. Genes that were expressed in freshly isolated BECs in at least 5-fold higher levels than in freshly isolated LECs with confidence level of P less than .0001 were included. MHC class II and MHC class II–related moieties form a prominent network in interaction plots generated with transcriptome data from freshly isolated BECs (dotted box, left). Different intensities of the red-colored symbols indicate higher (bright) or less pronounced (faint) overexpression in BECs versus LECs. The whole MHC class II cluster is lost in cultured ECs (dotted box, right). Gray symbols are used for transcripts that were at least 3-fold down-regulated in culture.
- Figure S2. Pan-EC–specific functional networks are maintained in culture (PDF, 64.9 KB) -
Pan-EC genes that are at least 5-fold higher expressed in both freshly isolated BECs and freshly isolated LECs than in non-ECs with a confidence level of P less than .0001 were included. A significant subcluster containing various moieties that play a critical role in EC adhesion and angiogenesis is present in both freshly isolated (left) and largely maintained in cultured ECs (right). White symbols indicate molecules that were not present in the original list of included pan-EC genes but were introduced by the software to complete the network. Gray symbols are used for transcripts that were at least 3-fold down-regulated in culture.
- Figure S3. In normal unperturbed tissue, BECs constitutively generate and present MHC class II peptide complexes (PDF, 79.4 KB) -
(A) Abs against CD1a (APC; x-axis, upper panel), CD14 (PC7; y-axis, upper panel), PDPN (PE; x-axis, lower panel), and VE-cadherin (PC5; y-axis, lower panel) were used to simultaneously resolve CD1a+ DDCs (blue dots), CD14+ DDCs (orange dots), BECs (red dots), and LECs (green dots) in freshly prepared dermal cell suspensions. (A-B) The 4 individual cell populations were gated and analyzed in the fifth dimension for cell-surface expression of MHC class II using FITC-labeled mAbs recognizing all HLA-DR (Tü36) or HLA-DP molecules, or reacting with peptide-loaded HLA-DR (L243) only (C). (D) Cells were permeabilized before immunostaining with FITC-labeled mAbs recognizing the class II invariant chain fragment CLIP, or the C-terminus or N-terminus of invariant chain. (C-D) Histograms indicate immunoreactivity of BECs (red), LECs (green), CD1a+ DDCs (blue), and CD14+ DDCs (orange). Dotted vertical lines denote the upper limit of the reactivity with isotype-matched control mAbs.
- Table S1. Pan-endothelial marker genes (PDF, 28.4 KB) -
Listed are genes that are greater than 20-fold higher expressed in freshly isolated ECs than, on average, in freshly isolated non-ECs (fibroblasts, mast cells, CD14+ dermal dendritic cells, CD1a+ dermal dendritic cells, Langerhans cells, and keratinocytes) with a confidence level of P < 1 × 10–7. Gray-shaded fields contain pan-EC genes whose expression is at least 5-fold down-regulated in cell culture.
- Table S2. LEC-specific antigens (PDF, 56.4 KB) -
Listed are genes that are greater than 10-fold higher expressed in freshly isolated LECs than in freshly isolated BECs, with a confidence level of P < 1 × 10–4. Bold symbols indicate genes that are expressed in freshly isolated LECs selectively (> 10-fold higher expressed in freshly isolated LECs than, on average, in freshly isolated BECs and the other reference skin cell types, with a confidence level of P < 1 × 10–6). Gray-shaded fields contain BEC-specific genes whose expression is at least 5-fold down-regulated in cell culture.
- Table S3. BEC-specific antigens (PDF, 62.7 KB) -
Listed are genes that are greater than 10-fold higher expressed in freshly isolated BECs than in freshly isolated LECs with a confidence level of P < 1 × 10–4. Bold symbols indicate genes that are expressed in freshly isolated BECs selectively (> 10-fold higher expressed in freshly isolated BECs than, on average, in freshly isolated LECs and the other reference skin cell types, with a confidence level of P < 1 × 10–6). Gray-shaded fields contain BEC-specific genes whose expression is at least 5-fold down-regulated in cell culture.
- Table S4. RMA normalized microarray data (XLS, 14.9 MB)
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