Western blot

After stromal cell culture using LNs from Atg5^ΔProx1 and Atg5^WT mice, LECs were sorted by flow cytometry (Astrios sorter; Beckman Coulter) and cultured (100,000 cells/well) in 12-well plates coated as before. At cell confluence, LECs were starved (HBSS) for 4 h to induce autophagy. Proteins from LECs (starved or not) were extracted using a lysis buffer (Tris, pH 8, 150 mM, NaCl 50 mM, and 1% NP-40) with protease inhibitor (Millipore). Proteins were quantified, and 5 µg was warmed at 95°C for 5 min and migrated onto a 12% SDS-acrylamide gel. After transfer on a polyvinylidene difluoride membrane, a saturation step was performed with PBS containing 5% milk for 1 h at RT, and anti-LC3 (1/2,000e; Cell Signaling) or anti-SphK1 (1/1,000e; BIOSS USA) antibodies were incubated overnight at 4°C under agitation. After washing, the membrane was incubated with secondary antibodies, goat anti-rabbit–HRP (1/10,000e; Bio-Rad), or anti-mouse–HRP (1/5,000e; Santa Cruz) for 1 h at RT, and the HRP substrate (Millipore) was used to reveal the proteins. Protein expression levels were normalized to GAPDH control protein expression level.

Human iLECs (provided by T. Petrova, University of Lausanne, Lausanne, Switzerland; Norrmén et al., 2010) were seeded in a 12-well plate coated with human fibronectin in EGM-2 medium (Lonza; Ruwag) at a concentration of 100,000 cells in 2 ml medium. At confluence, cells were starved in HBSS for 4 h, and Western blots were performed as described above.