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G. Li, I. Navarro, D. L. Epstein, P. Gonzalez, P. Challa; The Role of the Forkhead Box O Genes in Lens Cell Culture Response to Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2278.
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© ARVO (1962-2015); The Authors (2016-present)
To identify the expression of the FOXO gene family in lens cell cultures and to evaluate potential roles they might play in response to oxidative stress.
Oxidative stress was induced by incubation of B3 lens epithelial cells in 40% oxygen and 5% oxygen condition was used as normal control. B3 cells were treated with resveratrol 25µM once a day for five days in both 5% and 40% oxygen and the same concentration of ethanol was used as vehicle control. Total protein was isolated and western blot performed using antibodies against FOXO1A, FOXO3A, and FOXO4 (Cell signaling Inc.). The production of intracellular reactive oxidative species (iROS) was assessed by staining cells with H2DCFDA and flow cytometry. Glutathione levels were measured using Glutathione Assay Kit (Bioassay Systems Inc.). Cellular senescence markers SA-beta-galactosidase activity (FDG) and autofluorescence (AF) were determined using a flow cytometer. In order to study the roles of FOXOs in the cellular response to oxidative damage, siRNAs of FOXO1A, FOXO3A and FOXO4 (Ambion Inc.) were transfected into B3 cells.
The expression of FOXO1A, FOXO3A, and FOXO4 were detected by both realtime RT-PCR and Western blot in B3 cells. The levels of iROS, FDG, AF were significantly increased in 40% oxygen. Resveratrol dramatically reduced the production of these iROS, FDG and AF, and significantly increased antioxidant glutathione production. The FOXOs siRNA significantly reduced their corresponding levels of messenger RNA.
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