Phospho-MFF (Ser172/Ser146) Antibody - #AF2365

Fig. 5: Mitochondrial dynamics in 3D migration. 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.

Fig. 6 The cAMP/PKA/AMPK signaling pathway contributes to caffeine-induced mitochondrial fission and mitochondrial distribution to lamellipodia region. HUVECs were incubated with vehicle or caffeine (50 μM) in the presence or absence of H89 (10 μM) or compound C (5 μM) for the indicated durations. a, b Representative mitochondria from HUVECs cultured for 6 h (n = 3). Scale bar, 25 μm. c–e Representative images and quantification of the lamellipodia extent (d; n = 10) and relative fluorescent intensity of mitochondria in the lamellipodia region (e; n = 5) in HUVECs cultured for 12 h. Scale bar, 25 μm. f, g Western blot analysis of p-MFF protein levels in HUVECs incubated with vehicle or caffeine (50 μM) in the presence of 8-Br-cAMP (200 μM), H89 (10 μM), or compound C (5 μM) for 2 h (n = 3). *P 

Figure 3. Effects of γ-ray irradiation on AMPK, DRP1 and MFF signaling and changes in PTPMT1 expression in iPSC-CMs. qPCR (A) and Western blotting (B) were used to determine the expression of AMPK, and the histogram shows the quantitative results of B (C). Western blotting was used to determine the expression of fission-related proteins (p-DRP-1 and p-MFF; D), and the quantitative results are shown (E). qPCR (F) and Western blotting (G) were used to determine the gene expression level of PTPMT1, and the quantitative results of G are shown (H). Data are expressed as the means ± SE, n = 3 (replicates). One-way ANOVA was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, vs. 0 Gy group; ##P < 0.01, ###P < 0.001, ####P < 0.0001, vs. 10 Gy group. iPSC-CMs, induced pluripotent stem cell-derived cardiomyocytes; p-DRP1, phosphorylated-dynamin-related protein 1; p-MFF, phosphorylated-mitochondrial fission factor; PTPMT1, protein tyrosine phosphatase, mitochondrial 1.

Fig. 6. MFF is activated by AMPK and regulates mitochondrial fission and mitophagy in dendrites during dendritic outgrowth. (A,B) Phosphorylation of AMPKα and its downstream effectors in neurons treated with either TTX+APV (6 h), 100 µM glutamate (5 and 10 min) or 2 mM AICAR (30 min and 1 h) at DIV5. Blots were probed with antibodies against total and p-Thr172 AMPKα, total and p-Ser146 MFF, total and p-Ser555 ULK1, and β-actin. (C,D) Relative amount of phosphorylated forms to total proteins of AMPKα, MFF and ULK1. Samples were taken from four to six independent experiments. **P

Figure 5: Mitochondrial dynamics in cells adopting different modes of migration in 3D. A) Upon AMPK activation (A769662 10 mM, 30 minutes) in A375P cells, western blot showing levels of AMPK and MFF phosphorylation (n=3). Quantification normalized by total AMPK and MFF, respectively. B) Cells seeded on a collagen I matrix. (Left) Representative images of mitochondrial network (Tom20, green), F-actin (red) and nucleus (Hoechst, blue) after A769662 treatment (10 mM, 24 hours). Scale bar = 5 mm. (Right) Quantification of mitochondrial branches per cell (n=3). C) Western blot of AMPK and MFF phosphorylation levels after AMPK knock-down (siPRKAA1/2) in A375M2 cells (n=3). Quantification normalized by total MFF. D) Cells seeded on a collagen I matrix. (Left) Representative images of mitochondrial network (Tom20, green), F-actin (red) and nucleus (Hoechst, blue) after AMPK knock-down. Scale bar = 5 mm. (Right) Quantification of mitochondrial branches per cell (n=3). E) Live cell imaging of mitochondria using MitoTracker Deep Red and tetramethylrhodamine, ethyl ester (TMRE) in a panel of cell lines transfected with LifeAct-GFP and seeded on a collagen I matrix. Scale bar = 10 mm. F) Magnification of HT1080 and A375M2 mitochondria stained with MitoTracker Deep Red segmented using Wolfram Mathematica software. G) Quantification of mitochondrial circularity (n=3). H) Quantification of mitochondrial branches per cell (n=3). I) Quantification of TMRE fluorescence intensity normalized by mitochondrial mass per cell, by immunofluorescence, as readout of mitochondrial activity (n>6 cells/condition). J) Cells seeded on a collagen I matrix. (Left) Representative images of mitochondrial network (Tom20, green), F-actin (red) and nucleus (Hoechst, blue) after DDR1 knock-down and Comp C treatment (2 mM, 24 hours). Scale bar = 5 mm. (Right) Quantification of mitochondrial branches per cell (n=3). Dot plots (B, D, G, H, J) show median with interquartile range (each dot represents a single cell). Box plots (I) show min to max. p value by unpaired t-test (B, D, H right), unpaired t-test comparing elongated-mesenchymal versus rounded-amoeboid cells(G, H left, I) and Kruskal-Wallis with Dunn’s multiple comparisons test (J). For all graphs, ns non-significant, **p