Fig. 6

Autophagy phenotype in CAFs. (A) Western blot analysis showing increased levels of an autophagy marker, LC3B in all CAFs examined compared to the RMFs, under a basal culture condition. (B) Autophagy flux analysis. Fibroblasts were nutrient starved (HBSS buffer) for 6 h with or without treatment with the autophagy inhibitor bafilomycin A1 (Baf; 100 nM). Additional increased in LC3B expression in response to starvation and the lysosomal inhibitors were observed in CAFs. (C) Autophagic flux was further assessed using a tandem fluorescent-tagged ptfl-LC3 plasmid (mRFP-EGFP-LC3). Confocal microscopy analysis was performed after 24 h of transfection. At this time point, RMF still showed extensive acidified autophagosomal structures (yellow merged punta), while both CAFs showed a much reduced levels of GFP-LC3, as reflected by the remaining red puncta in the merged images. Representative GFP-LC3, RFP-LC3 and overlay images are shown. Right panels are enlarged photographs from the boxed areas. Scale bar represents 10 μm