3-(1H-Benzo[d]imidazol-6-yl)-5-(4-fluorophenyl)-1,2,4-oxadiazole (DDO7232), a Novel Potent Nrf2/ARE Inducer, Ameliorates DSS-Induced Murine Colitis and Protects NCM460 Cells against Oxidative Stress via ERK1/2 Phosphorylation

Figure 2

DDO7232 promoted the translocation of Nrf2 into the nucleus. (a) Nrf2 accumulated in the nucleus with the treatment of DDO7232. Nuclear and cytoplasmic extracts of NCM460 cells were obtained for Western blot analysis after treatment with DDO7232 (20 μM) for different durations of time (0 to 24 h). β-Actin and histone were used as internal references for cytosolic and nuclear Nrf2, respectively. Densitometric analysis was used to determine the relative ratios of the Nrf2 protein in each fraction. Nuclear Nrf2 expression was normalized to histone expression, and cytosolic Nrf2 expression was normalized to β-actin expression. Data are expressed as the of three individual experiments. The data were analyzed using ImageJ 1.44p. (b) Immunofluorescence staining of Nrf2 and nuclei in NCM460 cells. The nuclei and Nrf2 were labeled with DAPI (blue) and rhodamine-labeled rabbit IgG antibody (red), respectively. The relative potency of DDO7232 at 5 μM and 20 μM for 12 h was assessed using the classical Nrf2 activator tBHQ as a positive control.

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