Fig. 3.

ECM stiffness regulates mitochondrial dynamics and biogenesis. (A–C) Mitochondrial morphology in MSCs on soft and stiff ECM on days 1 and 7. Representative STED images (top) and high magnification of white boxed regions (bottom) were shown (A), scale bars, 20 μm and 4 μm. Mitochondrial length (B, n = 30 for each group) and mitochondrial footprint (C, n = 30 for each group) were analyzed. (D) Left, representative images of classification of mitochondrial morphology, scale bars, 20 μm. Right, quantification of punctate, intermediate, and filamentous mitochondria (n = 10 for each group). (E) Mitochondrial probability map (MPM) was plotted for the accumulation of mitochondria in MSCs (n = 10 for each group). (F) Mito⁹⁵ values (95th percentile) were calculated (left to right, n = 12, 10, 11, 12). (G–J) Transmission electron micrographs of mitochondria. Representative images of mitochondria (G), scale bars, 500 nm. The average mitochondrial length (H, left to right, n = 74, 74, 79, 83), relative mitochondrial size (I, left to right, n = 120, 116, 79, 88), and relative mitochondrial area per ROI (J, left to right, n = 11, 14, 10, 13) were quantified. (K) The mtDNA copy number was detected by qPCR (n = 4 for each group). (L) Time-lapse frames of mitochondrial fission and fusion. Red arrow, fission event; yellow arrow, fusion event, scale bars, 15 μm. (M) Quantification of fission and fusion events per minute per cell and (N) mitochondrial velocity (n = 30 for each group). (O) Left, immunoblots for mitochondrial fission and fusion protein expression in MSCs on day 7. Right, quantification of MFN1, MFN2 and DRP1 protein expression (n = 8 for MFN1, 7 for MFN2, 11 for DRP1). Data from at least three independent experiments. All graphs showed mean ± s.d. Statistical significance was derived from Mann-Whitney test (B,C,F,H,I) or unpaired two-tailed Student's t-test (J,K,M−O).