Fig. 1.

ECM stiffness promotes osteogenic differentiation of MSCs with increased ATP production. (A) RUNX2 gene expression of MSCs on soft and stiff ECM for 1–7 days (n = 4 for each group). (B) Left, representative images of ALP staining in MSCs for 1–7 days, scale bars, 200 μm. Right, quantification of ALP intensity (left to right, n = 12, 12, 10, 14, 19, 15, 14, 13). (C) Normalized ATP levels of MSCs on days 1 and 7 (n = 3 for each group). (D) Oxygen consumption rate (OCR) was determined by Seahorse in MSCs on days 1 and 7 (n = 4 for each group). (E–I) Quantification of individual parameters for basal OCR (E), maximal OCR (F), ATP dependent respiration (G), spare OCR (H), and H⁺ leak-linked OCR (I). (J and K) Extracellular acidification rate (ECAR) of MSCs (J) and quantification of basal ECAR (K, n = 4 for each group). (L) Mitochondrial membrane potential (ΔΨm) of MSCs on soft and stiff ECM on days 1 and 7. Left, representative images of JC-1 staining in MSCs, scale bars, 10 μm. Right, the ratio of J-aggregates to monomers (n = 30 for each group). (M) Levels of ROS in MSCs. Left, representative images, scale bars, 25 μm. Right, quantification of intensity (left to right, n = 30, 30, 36, 30). (N) The gene expression levels of SOD2 (n = 5 for each group) and CAT (n = 5 for each group) in MSCs. Data from at least three independent experiments. All graphs showed mean ± s.d. Statistical significance was derived from unpaired two-tailed Student's t-test, except for ROS intensity d 1 in M (Mann-Whitney test).