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  Indian J Med Microbiol
 

Figure 6: The reactive oxygen species-dependent activation of AMP-activated protein kinase by ethanol extract of Citrus unshiu peel in MCF-7 cells. (a and b) The cells were treated with 300 mg/m of ethanol extract of Citrus unshiu peel for the indicated times (a) or pretreated with N-acetyl-L-cysteine (10 mM) for 1 h before ethanol extract of Citrus. unshiu peel treatment for 30 min, which was followed by collection (b). The medium was discarded, and the cells were incubated at 37°C in the dark for 20 min with a new culture medium, which contained 10 mM of 2′,7′-dichlorofluorescin diacetate. Reactive oxygen species generation was measured by a flow cytometer. The data are the means of the two different experiments. (c and d) The cells were pretreated with 10 mM of N-acetyl-L-cysteine for 1 h before being treated with 300 mg/m of ethanol extract of Citrus unshiu peel. (c) After 24 h of incubation, the cellular proteins were separated by sodium-dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to the membranes. The membranes were probed with the indicated antibodies, and the proteins were visualized using an enhanced chemiluminescence detection system. Actin was used as an internal control. Representative images of at least three independent experiments are shown. (d) The viability of cells was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The data are expressed as the mean ± standard deviation of three independent experiments (*P < 0.05 vs. untreated control; #P < 0.05 vs. ethanol extract of Citrus unshiu peel-treated cells)

Figure 6: The reactive oxygen species-dependent activation of AMP-activated protein kinase by ethanol extract of <i>Citrus unshiu</i> peel in MCF-7 cells. (a and b) The cells were treated with 300 mg/m of ethanol extract of <i>Citrus unshiu</i> peel for the indicated times (a) or pretreated with N-acetyl-L-cysteine (10 mM) for 1 h before ethanol extract of <i>Citrus</i>. <i>unshiu</i> peel treatment for 30 min, which was followed by collection (b). The medium was discarded, and the cells were incubated at 37°C in the dark for 20 min with a new culture medium, which contained 10 mM of 2′,7′-dichlorofluorescin diacetate. Reactive oxygen species generation was measured by a flow cytometer. The data are the means of the two different experiments. (c and d) The cells were pretreated with 10 mM of N-acetyl-L-cysteine for 1 h before being treated with 300 mg/m of ethanol extract of <i>Citrus unshiu</i> peel. (c) After 24 h of incubation, the cellular proteins were separated by sodium-dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to the membranes. The membranes were probed with the indicated antibodies, and the proteins were visualized using an enhanced chemiluminescence detection system. Actin was used as an internal control. Representative images of at least three independent experiments are shown. (d) The viability of cells was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The data are expressed as the mean ± standard deviation of three independent experiments (*<i>P</i> < 0.05 vs. untreated control; <sup>#</sup><i>P</i> < 0.05 vs. ethanol extract of <i>Citrus unshiu</i> peel-treated cells)