Af 100 20

The cells were reseeded into 6-well plates and synchronized for 12 hours at 60% confluence. Then, the medium was replaced with H-DMEM with 2% FBS and VEGFA (5 ng/ml) or TGF-β (5 ng/ml), and cells were harvested with TRIzol after 48 hours.The cytokines used in the study were animal-free recombinant human VEGF165 (Peprotech, AF-100-20) and animal-free recombinant human TGF-β (Peprotech, AF-100-21C).

Cells were cultured in U-shaped, nonadherent 96-well plates (150 μL, 5.0 × 10³ per well) in StemSpan-ACF medium (#09855, Stem Cell Technologies) supplemented with 20% BIT9500 serum replacement (#9500, Stem Cell Technologies), CC100 (#02690, Stem Cell Technologies) mix of cytokines, 50 ng/mL VEGF (#AF-100-20, PeproTech), and penicillin with streptomycin (P4333-100ML, Sigma Aldrich). Compositions of other tested media are shown in Supplementary Table 1. After 48 hours, 50 μL of fresh medium was added; then, 50 μL of medium was changed every other day. On day 6 of culture, cells were stimulated with 7.5, 15, or 30 μmol/L atorvastatin (#3776, Tocris), 7.5, 15, or 30 μmol/L resveratrol (R5010-100MG, Sigma Aldrich), 125, 250, or 500 μmol/L acetylsalicylic acid (A5376-100G, Sigma Aldrich), 2.5, 5.0, or 10 μmol/L sulforaphane (S441-5MG, Sigma Aldrich), or 1.25, 2.5, or 5.0 mmol/L metformin (#317240-5MG, Sigma Aldrich). Conditioned media were collected after 48 hours. The controls, nonconditioned media, contained the same concentrations of stimulants and were kept for 48 hours in a cell culture incubator in the same conditions as stimulated cells. Wash-out conditioned media were collected from stimulated cells after washing with PBS and reseeding on 96-well plates in the complete fresh medium. DMSO, which was used to dissolve all stimulants, was added to the control wells.