Methyl 3 2 4 2 adamantyl phenoxy acetyl amino 4 hydroxybenzoate

Integrin-blocking antibodies α₅β₁ (Millipore) and α_vβ₃ (Santa Cruz Biotechnology) were added to cells at 1 μg/ml in medium before seeding and after changing the medium at 1 μg/ml. MAPK inhibitors [FR180204 (ERK1/2), SP600125 (JNK), and SB202190 (p38); 6 μM; Calbiochem], cytoskeletal inhibitor [blebbistatin (1 μM) and Y27632 (2 μM); Calbiochem], BMP inhibitor (Noggin; 5 ng/ml; ProSpec), GSK-3 inhibitor (CHIR; 10 nM; Calbiochem), HIF1 inhibitor (methyl 3-[[2-[4-(2-adamantyl)phenoxy]acetyl]amino]-4-hydroxybenzoate; 10 nM; Santa Cruz Biotechnology), hydrocortisone (0.5 mM; Tocris), and HSS (0.1 mg/ml; Tocris) were added to the medium after cell seeding with the first medium change. For inhibition assays, B16F0s were cultured for 3 days with the inhibitors, washed twice with PBS, and then cultured with fresh medium without the inhibitors to prevent the addition of the drugs in the conditioned medium to hMVECs.
siRNAs for Jarid1B (ID: 75605; Trilencer-27 Mouse siRNA) or scrambled siRNAs were purchased from OriGene. Transfection was performed according to the vendor’s instructions. Lipofectamine 2000 was used for transfection (100 nM). As with inhibition assays, cells were washed twice with PBS at day 3 and cultured with fresh medium to be conditioned.