Fig. 3: Blockade of mitophagy with high-dose MNU is rescued by DFP in vitro, ex vivo, and in vivo.

a Experimental design used to examine the effects of MNU in retinal explants (ex vivo): retinas from mito-QC mice (n = 4 per group) were isolated and cultured with different MNU doses for 18 h. Mitophagy and retinal thickness were quantified in fixed retinal explants. b Representative images of mito-QC retinal explants treated with different doses of MNU: 100, 500 and 1000 μg/ml and quantification of mitophagy density levels (red dots/mm² retina) (c) and volume of ONL (µm³) (d). e Experimental design used to examine the effects of MNU in retinal ARPE-19 mito-QC. f % cells with and without DAPI in MNU-treated ARPE-19 cells measured by flow cytometry and viability quantification. g Representative images of ARPE-19 mito-QC cells treated with increasing doses of MNU (left-right) and quantification of mitophagy by (h) fluorescence microscopy and (i) flow cytometry. j–m Quantification of mitophagy by flow cytometry in vehicle- and MNU (500 µg/ml)-treated ARPE-19 mito-QC cells subjected to downregulation of BNIP3L/BNIP3, PINK1/PRKN, BNIP3L and BNIP3 genes (left to right). n Quantification of viability based on DAPI intensity by flow cytometry in ARPE-19 mito-QC cells with different treatments (with/without 1000 µg/ml MNU and/or with/without DFP) in basal (left) or BNIP3L/BNIP3 silencing (right) conditions. o Quantification of mitophagy by flow cytometry in ARPE-19 mito-QC cells in different treatment conditions (with/without 1000 µg/ml MNU and/or with/without DFP) (right), and representative histogram depicting cell populations (left). Dots represent individual mice or different cell experiments. All data are expressed as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. Two-tailed Student´s t-test (for normally distributed data) and non-parametric Mann-Whitney U test (for non-normally distributed data) were used for statistical analyses. Scale bars: 25 µm.