Fig. 5. Effect of D1 on PRX2 hyperoxidation and SRXN1 Induction.

(A) Inhibition of PRX2 hyperoxidation. ARPE-19 cells were plated at 10⁵ cells/cm² and incubated for 24 h. Then, the medium was exchanged for serum-free medium containing vehicle (DMSO) or D1 (20 μM), and the cells were incubated for an additional 24 h, after which H₂O₂ (1 mM) or vehicle was added to the cells for 4 h. Lower panel: Cell lysates (10 μg protein/lane) were subjected to SDS-PAGE and immunoblotted for PRX2-SO_2/3H. Upper panel: Analysis of PRX2-SO_2/3H normalized to total PRX2 by taking the ratio of their densitometric values on the immunoblots. *p < 0.05. Each experiment was repeated at least twice. Samples loaded onto the gel in each experiment were from duplicate wells of a 6-well plate. Membranes were probed with β-actin to insure equal loading. (B) Induction of SRXN1. For mRNA levels, total RNA was extracted from ARPE-19 cells after treatment for 24 h with vehicle or D1 (20 μM) in serum-free medium and subjected to RT-PCR for SRXN1 and β-actin. For protein levels, lysates were prepared from cells after treatment with vehicle or D1 (20 μM); 10 μg protein samples were loaded per lane and subjected to SDS-PAGE prior to immunoblotting. The experiments were repeated two times with similar results. Analysis of SRXN1 normalized to total β-actin by taking the ratio of their densitometric values on the immunoblots. *p < 0.05.