Fig. 4

MLT restores autophagy in cells undergoing long-term ex vivo passaging. (MLT-treated P2, P7, and P15 cells were termed P2 + MLT, P7 + MLT, and P15 + MLT, respectively). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 represent significant differences between the indicated columns, while NS represents no significant difference. a Presence of autophagosomes (yellow) and their matured form, autolysosomes (red), in mRFP-GFP-LC3 adenovirus-transfected cells with or without MLT treatment (top: representative confocal images, scale bar = 50 μm, ×200 magnification; bottom: quantitative analysis of the percentage of yellow puncta out of the total puncta based on confocal examination). b GFP-LC3 fluorescence levels in P15 and MLT-treated P15 cells before and after Baf treatment (top: representative flow cytometry graphs; bottom: quantitative analysis of the MFI of GFP–LC3). c LC3-II levels in P2 and P15 cells before and after Baf treatment (top: protein levels of LC3-II as determined by Western blot assay; bottom: quantitative analysis of cell LC3-II expression). d Expression of cell autophagy-related proteins in P2, P7, MLT-treated P7, P15, and MLT-treated P15 cells as determined by Western blot assay and quantitative analysis of Atg7, p62, and Beclin-1 expression