Su4312

Chemicals purchased from Cayman Chemical (Ann Arbor, MI) include 5-azacytidine, Bay 11-7085, camptothecin, D-ribofuranosylbenzimidazole, H-8 HCl, kenpaullone, 3,4-Methylenedioxy-β-nitrostyrene (MNS), niclosamide, PD 98059, and topotecan hydrochloride. Citral, DMSO, LY-294002, retinol, and SU 4312 were purchased from Sigma-Aldrich (St. Louis, MO). CD437, ER50891, HX531, and SR11237 were purchased from Torcris (Minneapolis, MN). AM580 was purchased from Enzo Life Sciences (Farmingdale, NY). Chemicals were prepared as stock solution aliquots in DMSO and stored in the vapor phase of liquid nitrogen. Chemicals were protected from extended exposure to light when being prepared and used in assays. The LOPAC was purchased from Sigma-Aldrich and stored following the protocols at the National Center for Advancing Translational Sciences (NCATS) ^{22 (link)}. The Cignal lentiviral reagent, which had a lentivirus concentration of 1.7 × 10⁷ transducing units (TU)/ml, was obtained from SABiosciences (Qiagen, Frederick, MD). The MTT cell viability assay kit was obtained from ATCC (Manassas, VA) and used following the manufacturer's instructions.

Cells were starved in serum-free medium overnight. Cells were treated with 200 μM Na₃VO₄ in MCDB 131 for 5 min at 37°C, then VEGF (100 ng/ml) in MCDB 131 for 5 minutes , or with GO (1 U/ml) or 500 μM H₂O₂ in MCDB 131 for 5 to 90 min at 37°C. In experiments using inhibitors, cells were pretreated with the indicated inhibitor for 2 hours at 37°C. Cells were lysed in modified radioimmunoprecipitation (mRIPA) buffer as described previously (47 (link)). For in vivo assays, mice were perfused with 10 ml of 200 μM Na₃VO₄ in serum free DMEM, then either 10 ml of VEGF (200ng/ml), 1 mM H₂O₂ or vehicle in serum-free DMEM. Total lungs were lysed in mRIPA (100 μl mRIPA/10mg lung), homogenized, and centrifuged to clear debris. The following inhibitors were used at the indicated concentrations: anti-VEGF antibody, 10 μg/ml (Genentech), SU4312, 20 μM (Sigma), PP2, 10 μM (Millipore), NAC, 0.2 to 4 mM for in vitro experiments, 5mg/ml for mouse experiments (Sigma).