VEGF165 was obtained from R&D Systems (R&D Systems, Minneapolis, MN). Endothelial Basal Medium 2 (EBM2) was purchased from Lonza (Walkersville, MD). Fetal bovine serum (FBS), antibiotics, trypsin-EDTA, phosphate buffered saline (PBS), and other reagents for cell culture were purchased from Invitrogen (Carlsbad, CA, USA). BET inhibitor JQ1, VEGFR2 inhibitor axitinib and PAK1 inhibitor IPA3 were obtained from Selleck Chemicals.β-actin antibody was purchased from Sigma (St. Louis, MO, USA). The VEGFR2, and phospho-VEGFR2 antibodies were purchased from Cell Signaling. The anti-phospho-PAK1, anti-PAK1, anti-phospho-eNOS and anti-eNOS antibodies were obtained from Abcam (Cambridge, MA, USA).
For in vitro cellular assays, JQ1, IPA3 and axitinib were diluted in dimethyl sulfoxide (DMSO), and 0.1% DMSO was used as control. For in vivo experiments, according to previous report27 (link), a stock of 100 mg/ ml JQ1 in DMSO was 20-fold diluted by dropwise addition of a 10% 2-hydroxypropyl-β- cyclodextrin carrier (Sigma) under vortexing, harvesting a 5 mg/ ml final solution. Mice were intraperitoneally or intradermally injected daily with freshly diluted JQ1 (50 mg/ kg/d) or a similar volume of carrier containing 5% DMSO.
For in vitro cellular assays, JQ1, IPA3 and axitinib were diluted in dimethyl sulfoxide (DMSO), and 0.1% DMSO was used as control. For in vivo experiments, according to previous report27 (link), a stock of 100 mg/ ml JQ1 in DMSO was 20-fold diluted by dropwise addition of a 10% 2-hydroxypropyl-β- cyclodextrin carrier (Sigma) under vortexing, harvesting a 5 mg/ ml final solution. Mice were intraperitoneally or intradermally injected daily with freshly diluted JQ1 (50 mg/ kg/d) or a similar volume of carrier containing 5% DMSO.
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