**2.1. Cell culture and oxidative stress treatment**

Retinal pigment epithelial cells (ARPE-19) were purchased from ATCC (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (FBS; 10%) and penicillin/streptomycin (1%) at 37°C in a humidified atmosphere of 5% CO2 in air as suggested by the manufacturer. Cells were used between passages 8–9.

Retinal progenitor cells (HRP) were kindly donated by Dr. Harold J. Sheeldo (University of North Texas Health Science Center) and were cultured at the same condition as ARPE-19 cells.

Prior to all experiments, confluent ARPE-19 cells were incubated with fresh medium for 12 h and washed with phosphate buffered saline (PBS) three times. ARPE-19 cells were incubated with an oxidant, *tert*-butyl hydroperoxide (*t*-BuOOH, 200 μM, Sigma-Aldrich, St.Louis, MO), in serum-free medium for 0.5, 1, 2, 4, 6, 8, 12, and 24 h and representative images were presented. After the treatment, medium was removed, cells were washed with PBS and harvested for future analysis. Cells were lysed for experiments, including, immunoprecipitation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE), Western blot analysis, immunocytochemistry, and mass spectrometry analysis.

To compare with other stress environment, ARPE-19 cells were incubated under intense light (210 μmoles/m2 /s photon flux; 7000 lx) for 1 h in serum-free media and analyzed by SDS-PAGE/Western blotting, or immunocytochemistry.

Lipids were extracted from ARPE-19 cells using cholorform/methanol (2:1, v/v) and organic solvent was evaporated under a gentle nitrogen stream and dissolved in chloroform for analysis by HPLC and mass spectrometry.

All experiments were repeated (*N* = 3–10 biological samples) with technical duplicate or triplicate. Statistical analysis was performed using StatView software and statistical significance was determined by variance (ANOVA) or unpaired Student's *t* test when appropriate.
