Fig 2: Inhibition of TLR4 receptor blocked apoptosis and mitochondrial oxidative damage induced by Prx. (A) Following pretreatment with TAK242 (1 µM) for 1 h, RAW264.7 cells were incubated with Eh-rPrx (5 µg/ml) for 24 h, then suspended in 500 µL of 1× binding buffer (Sigma-Aldrich, APOAF) at a concentration of 106 cells/ml. Following incubation at room temperature for 10 min, 5 µL of Annexin V FITC conjugate (Sigma-Aldrich, APOAF) and 10 µL of propidium iodide solution (Sigma-Aldrich, APOAF) were added to each suspension. Fluorescence of the cells was immediately determined by using a flow cytometer. In all groups, n = 3. Experiments were repeated three times. (B) Quantitative analysis of mitochondrial ROS MFI in RAW264.7 cells treated with rPrx. RAW264.7 cells were pretreated with TAK242 (1 µM) for 1 h or oATP (500 µM) for 2 h. Next, the cells were treated with 5 µg/mL of Eh-rPrx or Tris-HCl (pH 8.0) as the negative control. After 24 h or 36 h of treatment, the live cells were stained with MitoSOX (5 µM) for 10 min, and then with CFSE (2 µM) for counterstaining. The cells were immediately observed and quantified through high-content screening analysis. In all groups, n = 3. Experiments were repeated three times. (C) The fluorescence images of RAW264.7 cells after 36 h of rPrx treatment. Red fluorescence, MitoSOX™ Red; green fluorescence, CFSE; scale bar, 100 µm. In all groups, n = 3. Experiments were repeated three times. Statistical analysis was conducted by Student’s t-test, and data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ns, not significant.