Figure 3
From: Automated high-throughput high-content autophagy and mitophagy analysis platform
Quantification of autophagy structures before and after addition of mitophagy or autophagy modulators. (A) Representative fields of live Rosella-LC3 and fixed LC3 antibody. Scale bar, 20 µm. Contrast of displayed images was adjusted by saturating the bottom and the top 1% of all the pixels values. (B) Heatmap of scaled (0.0–2.0) category-mean normalized absolute frequency per well for autophagic structures for live Rosella and fixed LC3 antibody Rosella-LC3. Each line in the heatmap represents the averaged amount of vesicles observed of a well. Significance matrix of Kruskal-Wallis and Dunn’s multiple comparison test is shown below. (C) Autophagic-vacuoles are quantified as the sum of autophagosomes, early autolysosomes, and late autolysosomes. Dunnett’s multiple comparison of means was performed for all conditions with respect to the basal reference (ref). Standard deviations are shown. Each dot represents the sum of objects in one imaged field. Data represent three independent replicates. (D) Representative lysotracker staining. Lysosome mask on red perimeter. Contrast of the image was adjusted by saturating the bottom and the top 1% of all the pixels values. Scale bars indicate 20 µm. (E) Lysosome frequency and diameter in basal conditions. Figure significance levels are *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
