Fig. 4.

ECM stiffness regulates osteogenic differentiation of MSCs through mitochondrial dynamics. (A) Top, representative images of mitochondrial morphology in MSCs with knockdown of MFN1 (siMFN1) and MFN2 (siMFN2), scale bar, 20 μm; inset scale bar, 10 μm. Bottom, quantification of mitochondrial length (left to right, n = 16, 10, 15, 11, 12, 12). (B) Top, Representative images of ALP staining in MSCs with knockdown of MFN1 (siMFN1) and MFN2 (siMFN2) for 7 days, scale bars, 200 μm. Bottom, quantification of ALP intensity (left to right, n = 10, 15, 11, 11, 9, 11). (C) ΔΨm in MSCs with knockdown of MFN2 (siMFN2) on stiff and soft ECM on day 7. Top, representative images of JC-1 staining, scale bars, 10 μm. Bottom, the ratio of J-aggregate to monomers (n = 26, 19, 30, 23). (D) Levels of intracellular ATP in MSCs with knockdown of MFN2 (siMFN2) on days 1 and 7 (n = 4 for 1 d, 3 for 7 d). (E) Levels of ROS of MSCs with knockdown of MFN2 on days 1 and 7. Left, representative images, scale bars, 25 μm. Right, quantification of intensity (n = 31 for siCO 1 d, 41 for siMFN2 1 d, 30 for siCO 7 d, and siMFN2 7 d). (F) DRP1 puncta were detected in MSCs on days 1 and 7. Top, Representative images of DRP1 puncta. Scale bar, 20 μm; enlarged inset scale bar, 10 μm. Bottom, the number of total DRP1 puncta (left to right, n = 33, 38, 30, 30). (G) Top, representative images of ALP staining in MSCs with DRP1 inhibitor Mdivi-1 (25 μM), scale bars, 200 μm. Bottom, quantification of ALP intensity (left to right, n = 23, 24, 23, 25). Data from at least three independent experiments. All graphs showed mean ± s.d. Statistical significance was derived from Mann-Whitney test (F) or unpaired two-tailed Student's t-test (A–E,G).