Fig. 4

Effects of methylene blue (MB) on MSC in vitro cultures. Consequences of MB addition to cell cultures were evaluated by analyzing MSC expansion potential (relative changes were normalized to MSC cultures without MB): a cell division time, b cellular senescence (C₁₂FDG staining), and c cumulative population doubling (CPD). The effects of MB on cell metabolism were analyzed by d cell mitochondrial activity (resazurin reduction assay) and e mitochondrial membrane potential (JC-1 staining) (n = 3; values represent the mean ± SEM. *p < 0.05. f Western blot analysis visualized changes in protein levels of respiratory enzymes in MSC cultured with or without MB, and g mitonuclear protein ratio was quantified by dividing SDHB (nuclear-encoded gene) by MTCO1 (mitochondrial-encoded gene). h MB induced expression changes in mitochondrial biogenesis and i chaperones genes in weak and vigorous MSC cultures at late passages (n = 3; values represent the mean ± SEM. Gene expression was normalized to GAPDH and normalized to expression of MSC cultures without MB (dotted line). *p < 0.05