Figure 2. Experimental validation that blue light exposure of A2E-treated ARPE-19 cells induces HMGB1 upregulation and translocation. (A) An MTT assay was performed on RPE cells treated with different concentrations of A2E with or without blue light photosensitization. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, *** indicates a p value < 0.001, compared to the control, n=3. (B) FDA/PI staining of RPE cells after in vitro culture for 48 h with 10 μM A2E + blue light (10 min). Most living RPE cells were stained green by fluorescein diacetate (FDA); a few dead cells were stained red bypropidium iodide (PI). (C) Western blot analyses showed that HMGB1 protein expression was higher in 10μM A2E + blue light-treated cells compared to the control and also higher in the blue light treatment, as quantified by densitometry; the results are expressed as a ratio with β-actin. Data are presented as means ± SD; * indicates a p value < 0.05, ** indicates a p value < 0.01, n=3. (D) HMGB1 localization in RPE cells was assessed by confocal microscopy after 10μM A2E + blue light treatment. HMGB1 moved from the nucleus (arrow) to the cytoplasm (star) after 10μM A2E + blue light treatment. Nuclei are labelled with DAPI (blue); HMGB1 is stained green.