Ntributing toEpicardial-Derived Interstitial Cellsthe identification of signaling pathways Title Loaded From File related to cardiac interstitium homeostasis and cell surface Title Loaded From File molecular profiles that could be used to characterize and isolate subpopulations of epicardial-derived CFs. This, in turn, could be instrumental to identify the roles that different CICs play in response to heart damage (i.e. fibrosis or active ECM degradation). A great variety of essential questions related to the maturation and response of CICs to episodes of hypoxia or inflammation remain open, and extensive and systematic research is required to develop new strategies to minimize cardiac fibrotic disease.Figure S4 cEP behaviour on TG-fibrin matrices: proteolytic activity and sprouting. A. cEP spheroids show different proteolytic/sprouting responses when cultured in TG-BPM2 and TGVEGF fibrin matrices as compared to control experiments (regular fibrin). HUVEC cells are shown as internal control for VEGF activity. B. cEP7 spheroids were embedded into 18325633 a 3D fibrin matrix with TG-bound-BMP2 and -VEGF121 or soluble bFGF, Wnt3a, Wnt5a, and examined after 48 h. cEP sprouting quantification after the different treatments has been graphically presented. Scale bars: 100 mm. (EPS) Figure S5 cEP4 zymography and protease inhibitor assays. A. 10 SDS-PAGE gels with 1.5 mg/ml gelatin were used to run cell culture supernatants. Gelatin degradation (48 hours of zymographic reaction) is shown for media from cEP4, EPICs, and proper controls, including plain culture medium, plasmin and 24272870 supernatant from HT1080 cells (HT1080 is a fibrosarcoma line known to express MMPs after TPA phorbol ester treatment). B. After 24 h cEP4 cells cultured on fibrin gels degrade the substrate and aggregate at the bottom of the culture dish (left, asterisk). Treatment with aprotinin reduces proteolysis and cells remain in the surface of the fibrin gel (arrowheads). (EPS)Supporting InformationFigure S1 Coronary endothelial angioblasts/cells do not followepicardial outgrowth in vitro. A. CD31 whole mount immunohistochemistry labels early subepicardial coronary angioblasts and endothelial cells (arrowheads). This kind of cell is absent from epicardial cell outgrowths in E11.5 whole heart explants (please, refer to Fig. 2G,H). B-B9. Microdissection of E11.5 mouse hearts in cold trypsin allows for the manual isolation of embryonic epicardial cells. Note that after this mechanical extraction, CD31+ angioblasts/endothelial cells can be found in epicardial explants in vitro (green). C . VEGFR-2 immunohistochemistry identifies vascular endothelium (asterisk, green) and angioblasts (arrowheads, green) in E13.5 mouse embryo samples (C), while EPICs remain VEGFR-2-negative (D). E. EPICs are immunoreactive to smooth muscle-specific myosin antibodies (red cells, arrowheads). Scale bars: A,B,C = 100 mm; B9,D,E = 50 mm. (EPS)Figure S2 Quantification of a and cSMA expression in TGFbinduced EPIC cultures. Quantitative PCR confirms the increased expression of a- and c-SMA in TGFb1-treated EPICs (left). TGFb2-treated cultures show an increased expression of c-SMA but not a-SMA (p value,0.05). (EPS) Figure S3 Ephrin and Eph EPIC profiling. Expression of EphrinAcknowledgmentsWe thank Dr. F. Weber (University Hospital, Zurich, Switzerland) for ?providing TG-BMP2, Dr. Eric G. Neilson (Vanderbilt University School of Medicine, Nashville, TN, USA) for the kind gift of the anti-FSP-1 antibody and Sanjay Giany for English editing. The authors also want t.Ntributing toEpicardial-Derived Interstitial Cellsthe identification of signaling pathways related to cardiac interstitium homeostasis and cell surface molecular profiles that could be used to characterize and isolate subpopulations of epicardial-derived CFs. This, in turn, could be instrumental to identify the roles that different CICs play in response to heart damage (i.e. fibrosis or active ECM degradation). A great variety of essential questions related to the maturation and response of CICs to episodes of hypoxia or inflammation remain open, and extensive and systematic research is required to develop new strategies to minimize cardiac fibrotic disease.Figure S4 cEP behaviour on TG-fibrin matrices: proteolytic activity and sprouting. A. cEP spheroids show different proteolytic/sprouting responses when cultured in TG-BPM2 and TGVEGF fibrin matrices as compared to control experiments (regular fibrin). HUVEC cells are shown as internal control for VEGF activity. B. cEP7 spheroids were embedded into 18325633 a 3D fibrin matrix with TG-bound-BMP2 and -VEGF121 or soluble bFGF, Wnt3a, Wnt5a, and examined after 48 h. cEP sprouting quantification after the different treatments has been graphically presented. Scale bars: 100 mm. (EPS) Figure S5 cEP4 zymography and protease inhibitor assays. A. 10 SDS-PAGE gels with 1.5 mg/ml gelatin were used to run cell culture supernatants. Gelatin degradation (48 hours of zymographic reaction) is shown for media from cEP4, EPICs, and proper controls, including plain culture medium, plasmin and 24272870 supernatant from HT1080 cells (HT1080 is a fibrosarcoma line known to express MMPs after TPA phorbol ester treatment). B. After 24 h cEP4 cells cultured on fibrin gels degrade the substrate and aggregate at the bottom of the culture dish (left, asterisk). Treatment with aprotinin reduces proteolysis and cells remain in the surface of the fibrin gel (arrowheads). (EPS)Supporting InformationFigure S1 Coronary endothelial angioblasts/cells do not followepicardial outgrowth in vitro. A. CD31 whole mount immunohistochemistry labels early subepicardial coronary angioblasts and endothelial cells (arrowheads). This kind of cell is absent from epicardial cell outgrowths in E11.5 whole heart explants (please, refer to Fig. 2G,H). B-B9. Microdissection of E11.5 mouse hearts in cold trypsin allows for the manual isolation of embryonic epicardial cells. Note that after this mechanical extraction, CD31+ angioblasts/endothelial cells can be found in epicardial explants in vitro (green). C . VEGFR-2 immunohistochemistry identifies vascular endothelium (asterisk, green) and angioblasts (arrowheads, green) in E13.5 mouse embryo samples (C), while EPICs remain VEGFR-2-negative (D). E. EPICs are immunoreactive to smooth muscle-specific myosin antibodies (red cells, arrowheads). Scale bars: A,B,C = 100 mm; B9,D,E = 50 mm. (EPS)Figure S2 Quantification of a and cSMA expression in TGFbinduced EPIC cultures. Quantitative PCR confirms the increased expression of a- and c-SMA in TGFb1-treated EPICs (left). TGFb2-treated cultures show an increased expression of c-SMA but not a-SMA (p value,0.05). (EPS) Figure S3 Ephrin and Eph EPIC profiling. Expression of EphrinAcknowledgmentsWe thank Dr. F. Weber (University Hospital, Zurich, Switzerland) for ?providing TG-BMP2, Dr. Eric G. Neilson (Vanderbilt University School of Medicine, Nashville, TN, USA) for the kind gift of the anti-FSP-1 antibody and Sanjay Giany for English editing. The authors also want t.
