Volume 3, Issue 3, Pages 287-301.e3 (September 2016) Analysis of Body-wide Unfractionated Tissue Data to Identify a Core Human Endothelial Transcriptome  Lynn Marie Butler, Björn Mikael Hallström, Linn Fagerberg, Fredrik Pontén, Mathias Uhlén, Thomas Renné, Jacob Odeberg  Cell Systems  Volume 3, Issue 3, Pages 287-301.e3 (September 2016) DOI: 10.1016/j.cels.2016.08.001 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Systems 2016 3, 287-301.e3DOI: (10.1016/j.cels.2016.08.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 CLEC14A, vWF, and CD34 Transcript Quantities In Vivo Reflect the Degree of Tissue Vascularization (A) Mean FPKM values for c-type lectin domain family 14, member A (CLEC14A), von Willebrand factor (vWF), and CD34 (CD34) transcripts in bone marrow, pancreas, ovary, tonsil, salivary gland, appendix, spleen, thyroid gland, gallbladder, urinary bladder, heart muscle, and lung; n = 2–5 individual samples/organ (see Table S1). Data are mean ± SEM. Corresponding IHC images stained with primary antibodies against CLEC14A, vWF, and CD34 protein are shown on tissue sections from ovary, appendix, gall bladder, and lung. (B) Scatterplots showing correlations between mean CLEC14A, vWF, and CD34 FPKM values and the estimated mean EC percentage in the sequenced sample, determined by histological examination prior to processing. Tissue type represented by each symbol corresponds to that indicated on the x axis of (A). Pearson correlation and corresponding p values are shown in the top left of each scatterplot. See also Figure S1A. Scale bars, 100 μm. Cell Systems 2016 3, 287-301.e3DOI: (10.1016/j.cels.2016.08.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Correlation Values between the Reference Endothelial Cell Transcripts CLEC14A, vWF, CD34 and Proteins Described as EC Enriched in the Literature (A) RNA-seq data from 124 individual samples from 32 different human tissue types were used to generate Spearman pair wise correlation values between the EC reference transcripts CLEC14A, vWF, and CD34 and transcripts reported in the literature as EC enriched. (B) IHC images of salivary gland, gallbladder, and lung tissue stained for proteins encoded by HSPA12B, PECAM1, ENG, ESM1, LIPG, and EDF1. Corresponding scatterplots (right) show the correlation between mean FPKM values and mean EC percentage in selected sequenced tissue samples. Tissue type represented by each symbol corresponds to that indicated on the x axis of Figure 1A. Pearson correlations and corresponding p values are shown for each scatterplot. Scale bars, 50 μm. Cell Systems 2016 3, 287-301.e3DOI: (10.1016/j.cels.2016.08.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Summary of Endothelial Cell Reference Transcript Correlation Analysis Data RNA-seq data from 124 individual samples from 32 different human tissue types were used to generate pairwise correlation values between the EC reference transcripts CLEC14A, vWF, and CD34 and the other 20,073 detectable protein-coding genes. (A) 234 transcripts were identified as EC enriched and categorized as known (previously reported as EC expressed), unknown (not reported as EC expressed), or uncharacterized. The ten most highly correlated in each category are displayed (p < 0.001 in all cases). (B) Scatterplots showing the correlation between mean FPKM values for selected genes from each category and the mean EC percentage in the sequenced tissue sample, determined by histological examination prior to processing. Tissue type represented by each symbol corresponds to that indicated on the x axis of Figure 1A. Pearson correlation and corresponding p values are shown in the top left of each scatterplot. See also Table S3, tab 1. Cell Systems 2016 3, 287-301.e3DOI: (10.1016/j.cels.2016.08.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 EH-Domain Containing 2 Is a Pan Endothelial-Enriched Protein In Vivo (A) IHC staining of multiple tissue types using a primary antibody targeting EHD2. Scale bars, 100 μm. (B.i.) Plotted mean FPKM values for von Willebrand factor (vWF) and EHD2 transcripts in 124 individual samples from 32 different human tissue types. Data are represented as mean ± SEM. Corresponding IHC images from liver, kidney, adrenal gland, and ovary (denoted by dotted boxes) are displayed above. (B.ii.) Staining for EHD2 in (1) veins, (2) venules, and (3) capillaries of the heart muscle. Scale bars, 50 μm. Data are represented as mean ± SEM. See also Figure S2D. Cell Systems 2016 3, 287-301.e3DOI: (10.1016/j.cels.2016.08.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 LIM and Senescent Cell Antigen-like Domains 2 Is Pan Endothelial-Enriched Protein In Vivo (A) IHC staining of multiple tissue types using a primary antibody targeting LIMS2. Scale bars, 100 μm. (B.i.) Plotted mean FPKM values for von Willebrand factor (vWF) and LIMS2 transcripts in 124 individual samples from 32 different human tissue types. Data are represented as mean ± SEM. Corresponding IHC images from liver, kidney, small intestine, and prostate (denoted by dotted boxes) are displayed above. (B.ii.) Staining for LIMS2 expression in (1) veins, (2) venules, and (3) capillaries of the heart muscle. Scale bars, 50 μm. Data are represented as mean ± SEM. See also Figure S2E. Cell Systems 2016 3, 287-301.e3DOI: (10.1016/j.cels.2016.08.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Family with Sequence Similarity 110, Member D Is a Pan EC-Enriched Protein In Vivo (A) IHC staining of multiple tissue types using a primary antibody targeting FAM110D. Scale bars, 100 μm. (B.i.) Mean FPKM values for von Willebrand factor (vWF) and FAM110D transcripts in 124 individual samples from 32 different human tissue types. Data are represented as mean ± SEM. Corresponding IHC images from liver, kidney, skeletal muscle, and ovary (denoted by dotted boxes) are displayed above. (B.ii.) Staining for FAM110D in (1) veins, (2) arterioles, and (3) capillaries of the heart muscle. Scale bars, 50 μm. Data are represented as mean ± SEM. See also Figure S2F. Cell Systems 2016 3, 287-301.e3DOI: (10.1016/j.cels.2016.08.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Pan EC-Enriched Transcript Expression in Cultured ECs (A.i) RNA-seq data from first passage primary umbilical vein endothelial cells (HUVECs) were used to identify the proportion of detectable known, unknown, and uncharacterized pan EC-enriched transcripts (FPKM ≥1). (A.ii) RNA-seq data from 124 individual samples from 32 different human tissue types were used to calculate a mean FPKM expression value for each pan EC-enriched transcript, which was plotted against the corresponding mean transcript expression in HUVEC (n = 4). Green, red, and black points represent known, unknown, and uncharacterized transcripts, respectively. Pearson correlations and corresponding p values are shown in the lower right of each plot. (B) Stomach, liver, cerebral cortex, and liver tissue sections stained for proteins encoded by GIPC3 and KANK3, pan EC-enriched transcripts that could not be detected in first passage HUVEC (unknown and uncharacterized category, respectively). Cell Systems 2016 3, 287-301.e3DOI: (10.1016/j.cels.2016.08.001) Copyright © 2016 Elsevier Inc. Terms and Conditions