Journal: PLoS ONE
Article Title: Molecular action of isoflavone genistein in the human epithelial cell line HaCaT
Figure Lengend Snippet: Attenuation of the level of reactive oxygen species (ROS) by genistein. (A) Keratinocytes were stimulated respectively with a proinflammatory “cytokine mix” corresponding to a concentration of 2 ng/mL (ACT 2) or 5 ng/mL (ACT 5) in each compound of the mix and incubated with 10 mM N-acetyl-cysteine (NAC) (ACT 2 + NAC), 100 μM genistein (ACT 2 + GEN), 10 ng/mL TNF-α or 1 μg/mL LPS alone, and with 10 ng/mL TNF-α or 1 μg/mL LPS incubated with 100 μM genistein (TNF-α + GEN or LPS + GEN). DMSO-treated, unstimulated cells were used as the control (NACT). Additional control was used in the form of unstimulated cells treated with 100 μM genistein (GEN). Intracellular ROS levels were examined by a CellROX Deep Red Reagent and confocal fluorescence microscopy. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Results representative of three independent experiments (with scale bars 25 μm) are shown. (B) Analysis of ROS were additionally performed by fluorescent cell analyzer. The data are presented as the means ± standard deviation (SD) from three independent experiments. Significant differences ( p ≤ 0.05) between cell populations not expressing intracellular ROS (ROS [–]) and expressing intracellular ROS (ROS [+]) were observed for all tested conditions, except for LPS where cell numbers of ROS (-) and ROS (+) were comparable. Most important, statistically significant differences of p ≤ 0.05 within ROS groups are indicated with * for samples of TNF-α, TNF-α + GEN, and LPS versus NACT, with † for sample TNF-α + GEN with respect to TNF-α, while with ‡ for sample LPS + GEN referred to LPS. Statistical analysis was performed using ANOVA with Tukey’s HSD test.
Article Snippet: For further experiments, cells were stimulated with a combination of a proinflammatory “cytokine mix”: IL-1A, IL-17A, IL-22, oncostatin M (OSM), and tumor necrosis factor-α (TNF-α) (Gibco, Thermo Fisher Scientific, CA, USA) 2 ng/mL each or lipopolysaccharide (LPS, from Escherichia coli 055 :B5 , Sigma-Aldrich, St. Louis, USA) 1 μg/mL in the presence or absence of genistein for 24 hours [ ].
Techniques: Concentration Assay, Activated Clotting Time Assay, Incubation, Fluorescence, Microscopy, Staining, Standard Deviation, Expressing