Human vegf165

Sorted Flk1⁺ cells were plated on gelatin-coated dishes in the differentiation medium. For induction to an endothelial cell, differentiation medium supplemented with 100 ng/mL human VEGF₁₆₅ (R&D Systems, Minneapolis, MN) was used. The medium was replaced every 2 days. After a 4-day culture, cells were fixed with 4% PFA (Wako Pure Chemical, Osaka, Japan) for 20 minutes at 4°C.
Then, specimens were blocked with 3% goat serum (Vector Laboratories, Burlingame, CA) with PBS (GIBCO) for 30 minutes at room temperature, and the following primary antibodies were applied to the sections at 4°C overnight: rabbit polyclonal anti-α-SMA antibody (1 : 200; Santa Cruz Biotechnology Inc., Santa Cruz, CA) or rabbit polyclonal anti-PECAM antibody (1 : 200; Santa Cruz Biotechnology Inc.). After washing, the following secondary antibodies were loaded for 1 hour at room temperature in a dark box: Alexa Fluor 488-coupled goat anti-rabbit IgG antibody (1 : 200; Invitrogen) or Alexa Fluor 594-coupled goat anti-rabbit antibody (1 : 300; Invitrogen). Finally, nucleus staining was performed using DAPI (Merck, Tokyo, Japan). The stained sections were observed using a fluorescence microscope (BX51, Olympus Optical, Tokyo, Japan) and images were obtained by a CCD camera (DP70, Olympus Optical).

Antibodies: anti‐human LIF polyclonal antibody was from Sigma (CAT# L9277). Normal goat IgG isotype control was from R&D Systems (CAT# AB‐108‐C). Anti‐LIF monoclonal antibody D25 (Kim et al, 1992 (link)) and mouse IgG2A isotype control were from Genentech.
Small‐molecule inhibitors: Baricitinib (Apexbio Technology, CAT# A414150), cobimetinib (MedChemExpress, CAT# HY‐13064), BEZ235 (Selleckchem, CAT# S1409), Z‐VAD‐FMK (R&D Systems, CAT# FMK001), Z‐DEVD‐FMK (R&D Systems, CAT# FMK004), Q‐VD(OMe)‐OPh (Apexbio Technology, CAT# A8165), 5‐AIQ hydrochloride (Sigma, CAT# A7479), CA‐074 me (Calbiochem, CAT # 205531), CA‐074 (Tocris, CAT # 4863), and CAA0225 (Calbiochem, CAT# 219502).
Recombinant Proteins: human LIF (Sigma, CAT# SRP9001), human LIF (Biolegend, CAT# 593902), human PDGF‐AA (Peprotech, CAT# 100‐13A), human Peroxiredoxin 1 (Abcam, CAT# ab74172), human IL‐8 (Biolegend, CAT# 574202), and human VEGF 165 (R&D Systems, CAT# 293‐VE).

Timed pregnant CD-1 (Charles River) or FLK1-eGFP mice (Samuel Lunenfeld Research Institute, Canada) were euthanized on E10.5 or E11.5 (presence of vaginal plug was considered embryonic day 0.5) and lung buds with intact tracheas were cultured on Transwell permeable supports with DMEM + Penicillin 12.5 U/ml/Streptomycin 12.5 μg/ml (Gibco) ±10% fetal bovine serum in the bottom well. In a similar approach, we performed some experiments with E15.5 post caval lobes with tracheas instead of whole lungs since accurate quantitation of lung explants at this stage is too difficult. Explants were cultured for up to 72 hours at 37°C in sealed chambers (Billups-Rothenberg, CA) equilibrated to a humidified atmosphere of 5% CO2 with a defined oxygen concentration, balanced by nitrogen. Culture medium was replaced every 24 hours. In some experiments, human Vegf165 (R&D Systems), human Vegf121 (R&D Systems), anti-rat NRP1 antibody (R&D Systems) or GO6983 (PKC pan inhibitor, EMD Chemicals, NJ, USA) were added to the culture medium at concentrations of 50–100 ng/ml, 50 ng/ml, 10 micrograms/ml and 1–5 microM respectively as previously described
[3 (link),6 (link),15 (link),16 (link)]. All animal experiments were conducted under approved Institutional Animal Care and Use Committee guidelines at Yale University.