Abstract
Purpose: :
We have previously shown that oxidative stress (OS) can quantitatively regulate AP-1 gene expression in RPE cells. In the present study we explore novel strategies for quantifying the protective effects of antioxidants using transcription factor (TF) and antioxidant protein biomarkers in a high throughput, cell-based assay.
Methods: :
ARPE-19 cells were seeded in 96-well plates and grown to confluence in defined media to stabilize gene expression. Cells were treated with 100µM or 350µM H2O2 for up to 6 hours and AP-1 family, EGR1/2, HO-1, and β-actin proteins were detected using IRDye 800CW-labled antibody. Fixed cells were permeabilized, labeled, and scanned at 700nm and 800nm wavelengths using an Odyssey imager to quantify biomarker and control protein levels.
Results: :
We quantified the expression of the AP-1 transcription factors, EGR1/2, HO-1, and a β-actin control. Following 100µM and 350µM H2O2 OS, AP-1 protein FosB increased 13% and 23%, cFos increased 15% and 48%, ATF3 increased 18% and 35%, and JunB increased 28% and 27% respectively, compared with the control wells. These data correlated well with our previously published Western results showing a dose-dependent increase in mRNA and protein levels in response to OS.
Conclusions: :
The high throughout cell-based Western assays may be a useful method to quantify OS-induced gene expression in RPE cells. The 96-well plate format permits us to sample the expression of target proteins under different incubation and stress conditions, and to evaluate temporal changes in protein expression over time in a high throughput manner.
Keywords: oxidation/oxidative or free radical damage • gene/expression • protective mechanisms