Gene expression analysis

Cells (10⁵ cells/well) were seeded in 6‐well plates. Serum deprived cells were treated with various inhibitors or TGFβ1. Total RNA from treated cells was extracted with the TRI‐reagent according to manufacturer's instructions (Ambion, Austin, TX, USA) and reverse‐transcribed to cDNA using TaqMan high performance reverse transcription reagents (Applied Biosystems, Life Technologies) at 25°C for 10 min., 37°C for 2 hrs followed by 85°C for 5 sec. in a Thermal cycler (BioRad‐DNA Engine; Bio‐Rad, Gladesville, NSW, Australia). The quantitative real‐time PCR reactions were performed in a 7300 system (Applied Biosystems, Life Technologies) by using TaqMan Universal PCR master mix and pre‐designed gene specific probes and primer sets for Nox2 (Hs00166163_m1), Nox4 (Hs01558199_m1 and Mm00479246_m1), Nox1 (Hs002455589_m1), Nox5 (Hs00225846_m1), EP300 (Hs00914223_m1) and NOS3 (Hs00167166_m1). The cycle threshold (CT) values form all quantitative real‐time PCR experiments were analysed using ^ΔΔCT method. Data were normalized to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; human 4326317E and mouse 4352339E) and expressed as fold changes over that in control treatment group.