Fig. 4. Antioxidative effects of 6’-SL in RAW 264.7 cells.
(A) RAW 264.7 cells were treated with 1 µg/ml LPS, 100 and 200 µM of 6’-SL, 10 µM of SB203580, or 10 µM of LY294002 for 24 h. Nrf2 translocation was analyzed by immunostaining with Nrf2 antibody. (B) Quantitively bar graph indicates relative fold change in nucleus Nrf2 expression. For quantification, cells within the sample images were enumerated based on DAPI-positive nuclei. The number of nucleus Nrf2-positive cells was then divided by the total cell count in each image. (C) RAW 264.7 cells were treated with 100 and 200 µM of 6’-SL, 10 µM of SB203580, or 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 24 h. Protein expression levels of Nrf2 and β-tubulin were detected by Western blot. (D) RAW 264.7 cells were co-transfected with ARE-Luc and Renilla-Luc for 24 h. After transfection, cells were pretreated with 100 and 200 µM of 6’-SL, 10 µM of SB203580, and 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 12 h. ARE-Luc promoter activity was measured using dual-luciferase reporter assay. (E) RAW 264.7 cells were treated with 100 and 200 µM of 6’-SL, 10 µM of SB203580, or 10 µM of LY294002 for 1 h, followed by treatment with 1 µg/ml LPS for 12 h. Total RNA samples were subjected to qRT-PCR using HO-1 primer. Data are expressed as the mean ± SEM (n = 3). 6’-SL, 6’-sialyllactose; LPS, lipopolysaccharide; Nrf2, nuclear factor erythoid 2-related factor 2; DAPI, 4’,6-diamidino-2-phenylindole; ARE, antioxidant response element; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; HO-1, heme oxygenase-1. **p < 0.01 compared to the control group, #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the LPS-treated group.
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