Fig. 2. TLR2/4 gene expression is ROS-dependent and is abrogated by ROS inhibitors. PBMC isolated from three healthy individuals were cultured (10⁶ cells/mL/well) in triplicate wells of 12-well plates and incubated for 10h with H₂O₂ (10mM) or vehicle only (mock). Intracellular ROS activity was measured using DCFH-DA assay. (A) The representative data from 3 independent determinations show the enhanced intracellular ROS activity in H₂O₂- treated cells compared to mock (SI=18.13). In other experiments, PBMC were treated with ROS inhibitors/anti-oxidants as well as H₂O₂ for induction of oxidative stress. Total RNA was extracted, and TLR2/4 gene expression was assessed using real-time RT-PCR as described in materials and methods. The data (mean±SEM) obtained from 5 independent determinations with similar results show the reduced gene suppression of: (B) TLR2 in cells treated with apocynin+H₂O₂ (6.97±0.12 fold, P=0.008) or N-acetyl cysteine+H₂O₂ (4.2±0.23 fold, P=0.0001); and (C) TLR4 in cells treated with apocynin+H₂O₂ (3.00±0.27 fold, P<0.0001) or N-acetyl cysteine+H₂O₂ (3.6±0.21 fold, P=0.0001) compared to respective controls treated with H₂O₂ alone.