Quantitative RT-PCR

For quantitative RT-PCR experiments, cells were plated in 6-well plates at a density of 0.5x10⁶ cells/well and were either kept untreated or treated with 50 ng/ml IFNγ in presence or absence of inhibitors for 24 h prior to RNA isolation. Quantitative RT-PCR experiments were performed in three different cell cultures as described previously [27]. In brief, RNA was purified with E.Z.N.A Total RNA kit (Omega Bio-tek, Norcross, USA). RNA was DNAse I treated in-column during the purification process and 500 ng RNA were reverse transcribed using random hexamers and Maxima reverse transcriptase according to manufacturer’s instructions (Fisher Scientific, Hampton, USA). Quantitative PCR was conducted on a C1000 Thermal Cycler (Biorad, Hercules, USA) with 30 ng of reverse transcribed RNA and DyNAmo Flash SYBR Green qPCR mix (Thermo Scientific, Walham, USA) using the following mouse specific primers: NOX1 (Forward, 5′-AATGCCCAGGATCGAGGT-3′; Reverse, 5′-GATGGAAGCAAAGGGAGTGA-3′), NOX2 (Forward, 5′-CCCTTTGGTACAGCCAGTGAAGAT-3′; Reverse, 5′-CAATCCCGGCTCCCACTAACATCA-3′), NOX4 (Forward, 5′-GGATCACAGAAGGTCCCTAGCAG-3′; Reverse, 5′-GCGGCTACATGCACACCTGAGAA-3′), L7 (Forward, 5’-GAAGCTCATCTATGAGAAGGC-3’; Reverse, 5’-AAGACGAAGGAGCTGCAGAAC-3’). Primers for the ribosomal protein L7 RNA were used as a control.