Fig. 5: Mitochondrial dynamics in 3D migration. | Nature Communications

Fig. 5: Mitochondrial dynamics in 3D migration.

From: AMPK is a mechano-metabolic sensor linking cell adhesion and mitochondrial dynamics to Myosin-dependent cell migration

Fig. 5

a Upon AMPK activation (A769662 10 μM, 30 min) in A375P cells, western blot of the indicated proteins (n = 3). b (Left) Representative images of mitochondrial network (Tom20, green), F-actin (red) and nucleus (Hoechst, blue) after A769662 treatment (10 μM, 24 h). Scale bar = 5 μm. (Right) Quantification of mitochondrial branches per cell from Tom20 staining (10, 17 cells pooled from n = 3). c Western blot of the indicated proteins after AMPK knock-down in A375M2 cells (n = 3). d (Left) Representative images of mitochondrial network (Tom20 (green), F-actin (red) and nucleus (Hoechst, blue)) after AMPK knock-down. Scale bar = 5 μm. (Right) Quantification of mitochondrial branches per cell from Tom20 staining (14, 13 cells pooled from n = 3). e Live cell imaging of mitochondria using MitoTracker Deep Red of the indicated cell lines stably transfected with LifeAct-GFP. Bottom panel show MitoTracker Deep Red segmentation used for quantification. Scale bar = 10 μm. Quantification of mitochondrial branches per cell from Tom20 staining (12, 20, 14, 30 cells pooled from n = 3). f Representative images of TMRE (red) and mitoTracker Green (green). Quantification of TMRE fluorescence intensity per cell (15 cells pooled from n = 3). g (Left) Representative images of mitochondrial network (Tom20, green), F-actin (red) and nucleus (Hoechst, blue) after DDR1 knock-down and Comp C treatment (2 μM, 24 h). Scale bar = 5 μm. (Right) Quantification of mitochondrial branches per cell from Tom20 staining (11, 10, 20, 10 cells pooled from n = 3). Western blot quantifications normalized by each total protein (a, c). Cells seeded on a collagen I matrix (b, d, e, f, g). Dot plots (b, d, e, f, g) show median with interquartile range (each dot represents a single cell). p values were calculated using two-tailed tests (b, d–f). p value by unpaired t-test (b, d, e, f) and Kruskal–Wallis with Dunn’s multiple comparisons test (g). All n are indicative of independent experiments unless otherwise stated. Source data are provided as a Source Data file. See also Supplementary Fig. 6.

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