Table 1.
Primer sequences used to amplify cDNA for qRT-PCR.
Figure 1.
Effect of genistein and LPS on RAW 264.7 macrophage viability.
Cells were pretreated with genistein (0 µM to 100 µM) for 1 h, then incubated with or without 1 µg/mL LPS for 12 h, 24 h, or 48 h respectively. Cell viability was determined by MTT assay. Data are the mean ± S.E.M (n = 3 ) of three independent experiments.
Figure 2.
Inhibitory effect of genistein on TNF-α and IL-6 mRNA levels in LPS-treated RAW 264.7 macrophages.
Cells were pretreated with genistein (0.1 to 10 µM) for 1 h and then incubated with LPS (1 µg/mL) for 24 h. Cells were collected, and IL-6 and TNF-α mRNA levels were determined by qRT-PCR and normalized to β-actin. Each column represents the mean ± S.E.M of triplicate experiments. **P<0.01 vs. control, ##P<0.01, #P<0.05 vs. LPS alone.
Table 2.
Effect of genistein on TNF-α and IL-6 production in LPS-treated RAW 264.7 cells.
Figure 3.
Effect of genistein on AMPK activation in LPS-treated RAW 264.7 macrophages.
(A) Cells were pretreated with 1, 5, or 10 µM genistein for 1 h and then incubated with 1 µg/mL LPS for 24 h. (B) Cells were pretreated with 10 µM genistein for 1 h and then incubated with 1 µg/mL LPS for 0.25, 0.5, 4, or 24 h. Cell lysates were prepared and analyzed for AMPK and p-AMPK by western blotting. GAPDH was used as an internal control. Experiments were repeated three times, and representative blots are shown here.
Figure 4.
Effect of genistein on NF-κB activation in LPS-treated RAW 264.7 macrophages.
(A) Cells were pretreated with 1, 5,or 10 µM genistein for 1 h, and then incubated with 1 µg/mL LPS for 24 h. Cells were pretreated with 10 µM genistein for 1 h, and then incubated with 1 µg/mL LPS for 0.25, 0.5, 4, or 24 h. (B) Cell lysates were prepared and analyzed for IκB-α, p-IKKα/β, or GAPDH by western blotting. The nuclear fraction was collected for assessment of NF-κB p65 and histones. GAPDH or histone was used as an internal control. Experiments were repeated three times, and representative blots are shown here.
Table 3.
Effect of genistein, AMPK activator, and AMPK inhibitor on TNF-α and IL-6 production.
Figure 5.
AMPK activation decreases the inhibitory effect of genistein on NF-κB activation in LPS-treated RAW 264.7 macrophages.
(A) Cells were pretreated with or without 10 µM genistein or 1 mM AICAR for 1 h and then incubated with 1 µg/mL LPS for 24 h. (B) Cells were pretreated with or without 20 µM Compound C for 30 min, then with or without 10 µM genistein for 1 h and stimulated with LPS for 24 h. Cytoplasm and nuclear extracts were collected for determination of AMPK, p-AMPK, I-κBα, p-IKKα/β, GAPDH, p65, and histone by western blotting. GAPDH or histones was used as an internal control. Experiments were repeated three times, and representative blots are shown here.