Fig. 6

p62-dependent lysophagy is essential for the neuroprotective effect of UA. A Knockdown of mitophagy (PINK1, PARK2), lysophagy (UBE2QL1, SQSTM1) or bulk autophagy (ATG5) in ARPE-19 cells was achieved through targeted siRNA lipofection. Knockdown efficiency was validated using RT-qPCR. B Viability assessment by nuclear exclusion assay (propidium iodide, PI) in ARPE-19 cells treated for 24 h using flow cytometry, selective autophagy regulators were genetically downregulated as shown in A. C Representative images and quantification of immunostained p62⁺ puncta (green) in ARPE-19 cells, nuclei were counterstained with DAPI (blue). D Representative images and quantification of immunostained p62⁺ puncta (green) in whole eye cryosections, nuclei were counterstained with DAPI (blue). E Representative images of ARPE-19 cells expressing the tfGal3 reporter and transfected with either Scramble- or SQSTM1-targeting siRNA (SQSTM1-KD), 1 mM LLOMe was used as positive control. Permeabilized lysosomes (RFP⁺GFP⁺, gray) and lysophagic lysosomes (RFP⁺GFP⁻, magenta) can be observed, nuclei were counterstained with DAPI (blue). F Quantification of lysophagic lysosomes as shown in E. Scale bars, 25 μm. All data are expressed as the mean ± s.e.m. Dots represent independent experiments (A, B) or biological replicates from three independent experiments (C, D, F). P values were calculated using unpaired Student’s t-test (A), 1-way ANOVA with Šídák’s (C, D) or Fisher’s LSD (F) post-hoc test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns: not significant