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The IC50 of BJ-1301, α-TOH, and sunitinib with respect to receptor tyrosine kinases

Specific actions of VEGF and BJ-1301 in NOX2 protein expression. Protein extracts from HUVECs treated with indicated concentration of BJ-1301 were loaded on SDS PAGE together with His-tagged human NOX1 (NOX1-1359H, Creative Biomart, NY, USA ), His+T7-tagged human NOX2 (LSG25251, LifeSpan Biosciences, WA, USA), and GST-tagged human NOX-4 (NOX4-1336H, Creative Biomart, NY, USA) recombinant proteins. Each isoform of NOX proteins was detected by using anti-NOX1 (ab55831, Abcam, MA, USA), anti-NOX2 (ab129068, Abcam, MA, USA), or anti-NOX4 (ab109225, Abcam, MA, USA) antibody. The membranes were reprobed to get the immunoblot of His, T7 or GST protein using anti-His (A00186100, Genscript, NJ, USA), anti-T7 (ab97964, Abcam, MA, USA), or anti-GST (ab19256, Abcam, MA, USA) antibody, respectively.

The IC50 values of BJ-1301, SU4312, and α-TOH on ROS production induced by VEGF and TPA

Primer sequences used in the PCR experiments

The Absolute mRNA expression (Ct andã…¿Ct values*) of NOX isoforms and regulatory subunits in A549 and H1299 cells

Effects of BJ-1301 and α-TOH on cell viability, proliferation, and VEGF-induced tube formation. A, Effects of α-TOH or BJ-1301 on the viability of HUVECs and BAOECs were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. B, Effect of BJ-1301 alone treatment on HUVEC proliferation. C, HUVECs plated in a well coated with Matrigel basement membrane matrix, were treated with compounds in the presence of VEGF (20 ng/ml). The total tube length per field was measured using Image Inside software (Upper panel). Invasion of HUVECs was measured using VEGF-induced HUVEC migration in the Transwell assay (Lower panel). The bar graphs represent the mean {plus minus} S.E.M. of three independent experiments. *P

Comparison of ID50 of BJ-1301 and α-TOH in VEGF-induced angiogenesis in vivo