Fig. 5.

Assessment of mito-QC Counter’s specificity in hypoxia-induced mitophagy with Bafilomycin A1. (A) Representative widefield fluorescence images of ARPE-19 cells expressing the mito-QC reporter and stimulated with hypoxia (1 % O₂) for 42 h and treated with 50 nM Bafilomycin A1 for the last 6 h of treatment. The number of mitolysosomes was quantified from three independent experiments with >50 cells for experiment. Zoom insets show representative mitolysosomes in both the fluorescence image and mitophagy mask. White arrows highlight example of mitolysosomes. Scale bar = 10 μm. (B) Total number of mitolysosomes counted per cell. (C) Total number of mitolysosomes counted per cell area defined by the manual selection of ROIs. (D) Mitolysosomal area expressed in percent of the cell area. (E) Mean size of the mitolysosomes. Quantification was performed with the following parameters: Radius for smoothing images = 1; Ratio threshold = 0.6; Red channel threshold = Mean + 0 stdDev. Statistics were realised using two-way ANOVA followed by a Bonferroni post-hoc test. n = 3 independent experiments. Statistical significance is displayed as **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).