Fig 2: Candidate gene expressions that contribute to injury (A-O) in HK2 cells exposed to aristolochic acidI (40 µM) compared to unexposed cells (control) showed increased expressions of TLRs (2–4, 9) and inflammatory cytokines (TNFa, IL6, IL1B, IL18, TGFß and NLRP3)); Unpaired t-test (two-tailed); n = 12; N = 4; Heatmap of gene expressions (P) showing TLR9 as the most prominent gene overexpressed in HK2 cells upon AA exposure (40 µM) for 24 h

Fig 3: Role of RAGE and TLR9 in the interferon-ß production by HSV-1 infection of corneal endothelial cells. (A) HCEn cells were transfected by siRNA of RAGE or control siRNA, and they were assessed for cell surface expression of RAGE with or without HSV-1 infection (multiplicity of infection (MOI) 1. Plated cells were stained for the surface expression of RAGE, and 1300 cells in high powered field per group were assessed for total fluorescence intensity. Si RNA transfection abolished HSV-1-induced-RAGE expression. HCEn cells were infected by HSV-1 infection at MOI 1 and were assessed for the expression of the mRNA of interferon-ß at 12 h by real-time RT-PCR. The HSV-1 infection induced-interferon-ß mRNA were significantly impaired by the RAGE blockade using siRAGE. (B) HCEn cells were infected by HSV-1 and were assessed for interferon-ß production at 24 h by ELISA. HCEn cells stimulated with HSV-1 infection at MOI 1 induced interferon-ß which was significantly reduced by anti-RAGE antibody and abolished by TLR9 inhibitory oligonucleotide (2 µM). *: p < 0.01; **: p <0.001; n = 6.

Fig 4: Chronic oxidative stress causes translocation of mtDNA to the cytoplasm to bind to the ZBP1 receptor in differentiated ARPE-19 cells. Analysis of binary interaction between mtDNA and TFAM in response to (A) 30 mU/ml of GOx and (B) 300 µM H2O2 visualized with PLA at 1 h post treatment. Note the marked decrease DNA/TFAM interaction in cells treated with GOx, but not with H2O2. C, time-dependent changes in interactions between mtDNA and intracellular DNA receptors (ZBP1, Z-DNA-binding protein 1; cGAS, cyclic GMP-AMP synthase; NLRP3, NOD, LRR and pyrin domain-containing protein 3 inflammasome; TLR9, toll-like receptor 9) in cells treated with 30 mU/ml of GOx. Please note the early stable interaction occurs only between mtDNA and ZBP1, and there is extranuclear localization of the signal. Representative images of n = 3 are shown. Scale bar, 10 µm. D, Western blot analysis of ZBP1-HA expression at 48 h post transfection with control or pCAGGS-HA-hZBP1 in ARPE-19 cells. E, the amounts of mtDNA and nuDNA in ZBP1s' IPs in control and GOx-treated ARPE-19 cells. Data are based on n = 3 and are expressed as mean ± SD. **p < 0.01 vs. vector control based on one-way ANOVA multiple comparison tests.

Fig 5: Association of TLR9, RAGE, and HSV-1. (A) Association of HSV-1 and RAGE by a GFP-pull-down assay. HCEn cells were infected with HSV-1 at MOI of 50, and the cell lysates as input were pulled down by anti-GFP antibody. The proteins associated with GFP-HSV-1 were detected by SDS PAGE. SDS PAGE, gel stained by Coomassie Blue, is shown as the input, pull down, and flow through fraction (upper panel). To observe RAGE and TLR9, Western blot for each fraction was conducted for RAGE and TLR9 (lower panel). (B) Tubulin and GFP in each fraction are shown by Western blot.