Figure 6.

Agm effect on the transcription factor Nrf2 and antioxidant enzyme HO-1 expressions. For nuclear extracts BV-2 cells were seeded at a density of 4.0 × 10⁴ cells/cm². Cells were pretreated with Agm (100 µm) for 30 min and then stimulated with Lps (1 µg/mL) for additional 0.5 and 24 h (Western blot analysis and fluorescent microscopy) or 4 and 6 h (qPCR analysis). (a) Nuclear protein level of Nrf2 relative to lamin B protein content 0.5 h upon Lps stimulation. (b) HO-1 gene (Hmox1) expression relative to the expression of the Gapdh gene and, (c) protein level relative to β-actin. (d) Nrf2 Western blotting (relative to β-actin) 24 h after Lps administration in cell lysates—left; micrographs with immunofluorescence labeling against Nrf2 (red) and Hoechst fluorescence labeling (blue), plotted scale bar applies to all images—middle; mean per-pixel fluorescence intensity (MFI) of the Nrf2 subunit in the nucleus of BV-2 cells was measured in ~200 cells per experimental group and analyzed as described in Materials and Methods—right. Protein levels are expressed relative to the control group (fold change) ± SEM (n = 4, a representative blot is shown). Results were analyzed by two-way ANOVA followed by Bonferroni’s post hoc tests (Western blot and immunofluorescence analyses) or Kruskal–Wallis test followed by Dunn’s multiple comparisons tests (gene expression analysis). Significance level: * p < 0.05 compared with control group; # compared with Lps group.