Fig. 1. Oxidative stress promotes the expression of TLR2 and TLR4 in PBMC. Peripheral blood mononuclear cells (PBMC) isolated from healthy individuals were cultured (10⁶ cells/mL/well) in triplicate wells of 12-well plates and incubated for 10h with H₂O₂ (10mM) or vehicle (mock). Total RNA was extracted from harvested cells using RNeasy kit following the manufacturer's instructions and TLR2/4 gene expression was assessed using real-time RT-PCR as described in materials and methods. At the same time, harvested samples were also stained to determine TLR2/4 receptor expression by flow cytometry. The data (mean±SEM) obtained from 5 independent determinations with similar results show the increased: (A) mRNA expression of TLR2 (6.63±0.94 fold) and TLR4 (4.11±0.25 fold) (P<0.001); and (B) protein expression (MFI) of TLR2 (412.3±8.4) and TLR4 (374.0±3.1) (P<0.0001) in H₂O₂-treated PBMC compared to respective controls (mock).