Fig. 2

SI induces proteostasis defects in mice and ARPE-19 cells that are reversed by UA administration. A Representative images and quantification of ProteoStat assay (yellow) to detect protein aggregates in whole eye cryosections. Nuclei were counterstained with DAPI (blue). B Representative images and quantification of LC3⁺ autophagosomes (gray) in whole eye cryosections, nuclei were counterstained with DAPI (blue). C Representative images and quantification of 4-hydroxynonenal (4-HNE) levels (Fire LUT) in whole eye cryosections. D Human-derived ARPE-19 cells were treated with 20 mM SI and/or 100 µM UA for 24 h. Viability was evaluated by nuclear exclusion assay (DAPI) using flow cytometry. E Representative images and quantification of 4-hydroxynonenal (4-HNE) levels (Fire LUT) in ARPE-19 cells. F Diagram depicting the basis of MAP macroautophagy tandem fluorescent reporter assay. G Representative images of ARPE-19 cells expressing the MAP reporter, 100 nM Bafilomycin A1 (Baf-A1) and EBSS were used as negative and positive controls, respectively. Autophagosomes (mCherry⁺GFP⁺, gray) and autolysosomes (mCherry⁺GFP⁻, magenta) can be observed, nuclei were counterstained with DAPI (blue). H Quantification of the number of autophagosomes as shown in G. I Quantification of the number of autolysosomes as shown in G. J Autophagy efficiency is reported as the number of autolysosomes per autophagosome, indicative of autophagic flux. Scale bars, 50 μm (A, B, C, E) and 25 μm (G). All data are expressed as the mean ± s.e.m. Dots represent individual mice (A, B, C), independent experiments (E, D) or biological replicates from three independent experiments (H, I, J). P values were calculated using Kruskal–Wallis with Dunn’s (A, B), 1-way ANOVA with Šídák’s (C, E), Tukey’s (D) or Fisher’s LSD (H, I, J) post-hoc test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns: not significant