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Table S1. Primary and secondary antibodies used in western blot experiments.

Table S2. Primary and secondary antibodies used in immunocytochemistry and immunohistochemistry.

Figure S1 Culturing of intact whole blood vessel with differential perfusion. (A) Veins from human umbilical cord were isolated and incubated for 48 hrs. External solution contained isotonic medium containing FBS and growth factors. Internal solution was HUVEC medium containing vehicle or 20 μg/ml LPS. External and internal solution were not mixed during the experiments. Solutions were changed frequently. (B) Cultured blood vessel was dissected to extract a small portion of vessel wall to exposed ECs monolayers. Samples extracted were 150 mm2 approximately. Vessel wall structure was not altered. (C) Monolayer samples were placed in a coverslip and immunohistochemistry experiments were carried out. Then, samples were mounted and images were acquired using a Floid Cell Imaging Station (Life Technologies™).

Figure S2. (A) Changes in viability of ECs exposed to TGFβ1. ECs exposed to 0, 1, 2, 3, 4, 5 and 10 μg/ml TGFβ1 for 72 hrs, evaluated by means of (A) MTT assay and (B) propidium iodide (PI) incorporation assay. In (A), cell viability was expressed relative to the untreated (0 μg/ml TGFβ1) condition. In (B), cells incorporating PI (empty bars, PI+) denote cell death and PI-negative cells (filled bars, PI+) denote healthy cells. Statistical differences were assessed by a one-way anova (Kruskal–Wallis) followed by Dunn's post hoc test. *< 0.05 and **P < 0.01 against the untreated (0 μg/ml TGFβ1) condition. Graph bars show the mean ± S.D. (= 3).

Figure S3 Cellular distribution of proteins involved in TGFβ1-induced endothelial fibrosis. Representative images from the experiments of untreated (AH) or 5 ng/ml TGFβ1-treated (IP) ECs for 72 hrs. CD31 or VE-cadherin (red), and α-sma, FSP-1 or FN (green) were detected. The box depicted in A, C, E and G indicates the magnification shown in B, D, F and H respectively. Arrows indicate CD31 (B and F) or VE-Cadherin (D and H) labelling at the plasma membrane, whereas arrowheads indicate α-sma (B), FSP-1 (D) or FN (F and H) staining, indicating basal expression of fibrotic markers (B and D) or ECM proteins (F and H). The box depicted in J, L, N and P indicates the magnification shown in I, K, M and O respectively. Arrows indicate α-sma (J), FSP-1 (L) or FN (N and P) labelling in plasma membrane, whereas arrowheads indicate CD31 (J and N) or VE-Cadherin (L and P) staining from residual endothelial marker expression indicating EndMT. Nuclei were stained using DAPI. Bar scale represents 10 μm.

Figure S4. Unspecific staining in immunocytochemical experiments when primary antibodies were omitted. Experiments performed in the presence (+) or the absence (−) of primary antibodies against: CD31 (A and B), VE-cadherin (C and D), α-sma (E and F), fibronectin (G and H) and type III collagen (I and J). Arrows depicted unspecific staining. Nuclei were stained using DAPI. Bars scale represents 10 μm.

Figure S5. Integrity of the endothelial monolayer from intact whole blood vessels after 0 or 48 hrs of perfusion. (AD) CD31 detection was performed in the endothelial monolayer from whole blood vessels perfused for 0 hr (A) or 48 hrs (C) in vehicle-perfused vessels. B and D show magnification of the box depicted in A and C respectively. Arrows indicate CD31 labelling at the plasma membrane. No significant changes were detected at 0 or 48 hrs of perfusion. Nuclei were stained using Hoechst. Bar scale represents 50 μm. (E and F) Structural integrity of endothelial monolayer from blood vessels was evaluated by haematoxylin eosin staining. Transversal slides from vehicle-perfused vessels incubated for 0 hr (A) or 48 hrs (B). Arrows indicate endothelial cells. Bar scale represents 50 μm.

Figure S6. Unspecific staining in immunohistochemical experiments when primary antibodies were omitted. Experiments performed in the presence (+) or absence (−) of primary antibodies against the following proteins: CD31 (A and B, in vehicle-perfused vessels), fibronectin (C and D, in LPS-perfused vessels) and type III collagen (E and F, in LPS-treated cells). Bar scale represents 50 μm.

Figure S7. ALK5 expression down-regulation by siRNA. Endothelial cells were transfected with a specific siRNA against human isoform of ALK5. (A). Representative images from western blot experiments performed for the detection of ALK5 in cells transfected with a siRNA against ALK5 (siALK5) or a non-targeting siRNA (siCTRL). (B) Densitometric analyses from several experiments, as shown in (A). Protein levels were normalized against tubulin, and the data are expressed relative to cells transfected with siCTRL condition. Statistical differences were assessed by student's t-test (Mann–Whitney). ***< 0.001. Graph bars show the mean ± S.D. (= 3).

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