Fig. 3. OS-induced DNA damage and cytosolic leakage provoke retinal cell inflammation.

Immunofluorescence (IF) staining for dsDNA A and dsDNA/rhodopsin B in retina. The DNA was counterstained with DAPI. Scale bar: 200 μM. C Schematic diagram for preparation of nuclei- and mitochondria-free retinal cytosol. D WB showing separation of retina nuclear and cytosolic fractions. E q-PCR analysis of cytosolic nuclear and mitochondrial (Mito) DNA. F, G IF staining in 661W cells using the indicated antibodies. Arrows: cytosolic DNA with positive γH2Ax labeling. Scale bar: 5 μM. WB showing retina proteins at the indicated time points (H) or 3-day (I) post SI injection. J WB showing indicated proteins in 661W cells. Cells were treated with or without Ara-C (10 μM) and collected at the indicated time points. K, L qRT-PCR analysis showing gene expression in ARPE cells. Cells were treated with DMSO or NAC (1 mM) or MitoQ (1 μM) for 24 h before collection. M, N WB analysis showing protein expression. M 661W cells were untreated (Ctrl) or, transfected with lipofectamine or linearized GFP-C3 plasmid (1 ug/ml) and harvested at the indicated time points. N ARPE cells were transfected with lipofectamine (Ctrl), or genomic DNA isolated from normal (nor-gDNA) or H₂O₂-exposred (H₂O₂-gDNA) ARPE or 661W cells (2 ug/ml) and collected 16 h post transfection. O qRT-PCR analysis showing indicated genes in ARPE cells transfected with or without ARPE genomic DNA as described as Fig. 6N.