Fig. 7

Increase in autophagic flux in Ras-NIH 3 T3 cells. A Immunoblotting was performed to measure LC3 conversion in cells treated with or without 30 μM CQ for 24 h. The values below each band represent autophagic flux as the difference in the amount of LC3-II normalized to loading control, β-actin, between the CQ-treated and untreated groups. B Protein expression levels of p62 were measured by immunoblot analysis. Representative images are presented along with β-actin as an internal loading control. The indicated values below each band represent normalized ratio of phosphorylated protein to total protein. The p62 protein level was normalized to β-actin and was shown as relative value below each band. The intensity value observed in control of NIH 3 T3 cells was defined as 1.0. C Autophagic flux was determined using ptfLC3 imaging assays. Representative images are shown in the upper panel. Yellow puncta are indicators of autophagosomes, whereas red puncta are indicative of autolysosomes in merged image. In lower panel, the average numbers of yellow and red puncta/cell were shown as a bar graph. Data are expressed as mean ± SD of at least 20 different cells. D Colocalization analysis of LC3 and Lysotracker. The cells were incubated in serum-free media containing 50 nM Lysotracker (red) stained with Lysotracker (red). Endogenous LC3 was visualized with the anti-LC3 antibody (green). Overlapping analysis was performed using the RG2B Colocalization plugin in ImageJ. Bar, 10 μm. Lower panel: LC3 puncta that colocalized with Lysotracker were counted in 50 cells using the JACoP plugin in ImageJ. The data shown are the average and standard deviation of ten randomly selected images. **P < 0.01 compared to NIH 3 T3 cells. E The phosphorylation of MEK, ERK, Akt, and mTOR was detected through immunoblotting. The indicated values below each band represent normalized ratio of phosphorylated protein to total protein