Figure 3. Advanced glycation end products–BSA (AGE-BSA) treatment results in the release of high-mobility group box protein 1 (HMGB1) from retinal ganglion cell (RGC)-5 cells. A: HMGB1 in the culture media after treatment of RGC-5 cells with 200 μg/ml of AGE-BSA or BSA alone was detected by western blot analysis. The release of HMGB1 into the media reached significance at 3 h after treatment with AGE-BSA, compared to the BSA control (n=4, * p<0.05; A.U.represents arbitrary unit). B: XTT test and (C) lactate dehydrogenase (LDH) assay conducted after incubation of RGC-5 cells with 200 μg/ml of AGE-BSA or BSA alone for 24 h. No significant difference was found between groups (n=6, p=0.515 for XTT and p=0.916 for the LDH test). D: RAGE and (E) TLR4 protein levels were assayed with western blot analysis. The levels of RAGE in the RGC-5 were not different between groups over time. However, a mild but significant increase in the TLR4 protein was detected after AGE-BSA treatment at 24 h (n=5, p=0.010).