A quantitative colorimetric assay employing dimethylthiazol (MTT assay; ATCC) was used to measure HPASMC proliferation. Briefly, following exposure to control or hypoxic conditions with or without treatment with rosiglitazone, HPASMC were incubated with the MTT reagent for 4 h. The mitochondrial reductase in living cells reduces Enzalutamide mw MTT to purple formazan, which is detected by spectrophotometry. Samples were analyzed using an ELISA plate reader at a wavelength of 570 nm. The values from treated cells were normalized to values from corresponding control cells. RNA isolation, reverse transcription, and quantitative PCR. Total RNA was isolated from cultured HPASMC using RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions, and RNA was quantified by NanoDrop spectrophotometry (Thermo Scientific, Wilmington, DE).
cDNA was prepared using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Quantitative PCR was performed to assess expression of Nox4 and GAPDH using primers that were designed based on human mRNA sequences. Nox4 primers were forward 5��-ggtta aacac ctctg cctgt tc-3�� and reverse 5��-cttgg aacct tctgt gatcc tc-3��, and GAPDH primers were forward 5��-cctgt tcgac agtca gccg-3�� and reverse 5��-cgacc aaatc cgttg actcc-3��. Real-time PCR was performed using iQ SYBR Green Supermix and the iCycler Real-Time PCR Detection System (Bio-Rad). Amplicon expression in each sample was normalized to its GAPDH RNA content. The relative abundance of target mRNA in each sample was calculated using ����CT methods as described by Applied Biosystems (User Bulletin no.
2). Western blot analysis. HPASMC were isolated, and equivalent amounts of protein were resolved by SDS-PAGE and immunoblotted with Nox4 rabbit polyclonal antibody (provided by Dr. David Lambeth, Emory University) or CDK4 rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) as previously reported (39). Relative levels of immunoreactive proteins were quantified using the ChemiDoc XRS imaging system and Quantity One software (Bio-Rad). Nox4 reporter construct, transient transfection techniques, and luciferase assays. The human Nox4 promoter spans nucleotides ?718 to +3 of the Nox4 gene locus relative to the position (+1) of the initiation methionine for the Nox4 open reading frame (PubMed Gene ID 50507).
The sequence was amplified from human genomic bacterial artificial chromosome clone RP11-735I13 (BACPAC Resources, Oakland, CA) by PCR using the following 5�� and 3�� primers: 5��-aacct cgagt cccct agagc cccta agaa-3�� and 5��-ggtaa gctta ggacc gaggg tcaaa gact-3��, respectively. The resulting PCR product was digested with Hind III and XhoI, inserted into the pGL4.10-basic luciferase GSK-3 reporter vector (Promega, Madison, WI), and confirmed by automated sequencing.