(B) Representative images of microvascular tubules in 7

(B) Representative images of microvascular tubules in 7.5wt% and 15wt% hydrogels after 14 days of tradition. peptide did not have a significant effect. Scale pub = 500 m.(TIF) pone.0244243.s002.tif (402K) GUID:?8C9FF1C5-47DA-4056-BB35-C02A2E6DCE8C S3 Fig: Secalciferol MVECs attach to PEG hydrogels containing RGD peptides. Representative images of MVECs cultured on non-degradable PEG hydrogels comprising 0 or 1 mM of RGD-MI peptide for 24 hrs. MVECs cultured on hydrogels comprising 1 mM RGD were adherent. Hydrogels lacking RGD did not promote cell adhesion, with MVECs observed to be rounded and clustered. Scale pub = 200 m.(TIF) pone.0244243.s003.tif (178K) GUID:?F4A1B6EE-3973-439E-9A76-30D73B9F5643 S4 Fig: MVEC viability is usually decreased over time in PEG-based hydrogels. Representative live/lifeless images MVEC monocultures in 7.5wt% hydrogels with 3 mM RGD, 3 mM YIGSR, and 3 mM laminin peptide cocktail after 1 Secalciferol and 7 days of tradition. Green shows live cell body and red shows nuclei in necrotic cells. Level pub = 100 m.(TIF) pone.0244243.s004.tif (249K) GUID:?BC59A4CB-88D6-43EA-9681-8FB47A107975 S5 Fig: Lumen structure formation in co-cultures after 28 days. Representative images MVEC microvessels in (A) 3:1 and (B) 3:3 co-cultures in 7.5wt% hydrogels with 3 mM RGD after Secalciferol 28 days of tradition. Z-stack cross-sections demonstrate the formation of a hollow lumen structure in 3:3 co-cultures. MVECs are depicted by CD31 (magenta) and nuclei are counterstained with Hoechst 33258 (cyan). Level pub = 50 m.(TIF) pone.0244243.s005.tif (742K) GUID:?A723120B-46FD-4EAC-B04C-9B6144DC00E6 S6 Fig: AoAF proliferate in both mono- and co-cultures. Assessment of the number of AoAFs per mm3 in Secalciferol (A) 3:3 co-cultures vs. 0:3 AoAF monocultures and (B) 3:1 co-cultures vs. 0:1 AoAF monocultures over time in 7.5wt% hydrogel with 3 mM RGD.(TIF) pone.0244243.s006.tif (142K) GUID:?5CB9C8A5-C95C-44BD-B109-25B9F796BBA6 S7 Fig: AoAFs and MVECs secrete basement membrane proteins in 2D culture. Monocultures of MVECs and AoAFs create collagen type IV (green) and laminin (green) on TCPS. F-actin is definitely stained with phalloidin-568 (gray) and nuclei are counterstained with Hoechst 33258 (blue). Level pub = 100 m.(TIF) pone.0244243.s007.tif (3.1M) GUID:?0E26A08A-186E-4C40-AF64-57D591CACC8E S8 Fig: Viability of MVECs is usually impaired when direct contact with AoAFs is restricted. (A) Representative live/dead images of MVECs in degradable PEG-based hydrogel (7.5wt% hydrogels with 3 mM RGD) cultured in either co-culture medium (1:1 EGM2-MV) or AoAF conditioned medium. (B) Representative live/dead images of MVECs and AoAFs in the restricted layered hydrogel system after 1 and 7 days of tradition. MVECs were encapsulated inside a coating of degradable PEG-based hydrogel (7.5wt% hydrogels with 3 mM RGD), while AoAFs were encapsulated inside a coating of non-degradable PEG-based hydrogel (10wt% hydrogels with 3 mM RGD). (C) In the unrestricted layered hydrogel system, MVECs were 1st encapsulated inside a coating of degradable PEG-based hydrogel, followed by encapsulation of AoAFs in a second coating of degradable PEG-based hydrogels. Green shows live cell body and red shows nuclei in necrotic cells. Level pub = 100 m.(TIF) ETS2 pone.0244243.s008.tif (864K) GUID:?A1835299-83C0-4444-8F9D-BCAB0F74287A S9 Fig: AoAFs and MVECs express TGF-1 and ALK5. Representative images of TGF-1 (green) and ALK5 (green) in AoAFs and MVECs cultured on TCPS. F-actin is definitely stained with phalloidin-568 (reddish) and nuclei are counterstained Secalciferol with Hoechst 33258 (blue). Level pub = 100 m.(TIF) pone.0244243.s009.tif (1.9M) GUID:?6E3D3EF4-D34E-4915-B9CA-5397A876280F S10 Fig: Inhibition of ALK5 reduces the nuclear localization of SMAD-2/3 while increasing cell proliferation. (A) Representative images of SMAD-2/3 (green) localization in AoAFs and MVECs cultured on TCPS, following treatment with either 0.5 M A83-01 (ALK5 inhibitor) or DMSO (control) for 72 hrs. F-actin is definitely stained with phalloidin-568 (reddish) and nuclei are counterstained with Hoechst 33258 (blue). Level pub = 100 m. (B) Normalized AoAF and MVEC proliferation following treatment with either 0.5 M.