Fig. 4: MT acetylation levels determine autophagosome positioning and autophagy flux.
A Immunostaining for LC3B and acetylated (Ac-) α-tubulin of p27+/+ MEFs glucose starved for 48 h (upper panel) and treated with 50 µM CQ for the last 2 h (lower panel). Cells were classified into three categories in function of their microtubule (MT) acetylation pattern: low MT acetylation; perinuclear acetylated MT and hyperacetylated MT. In each population, LC3B+ vesicles were analyzed to determine if there is a correlation between MT acetylation status and vesicle positioning. Scale bars are 50 µm. B Mean percentage of cells with distinct LC3B distribution patterns (Randomly distributed, less than 10 LC3B puncta and perinuclear LC3B puncta) in each MT acetylation pattern described in (A). The graph shows means ± SEM from n = 2 independent experiments. At least 65 cells were analyzed per experiment. The number of LC3B puncta was determined using Find maxima function in ImageJ. C Autophagy flux (high or low) was determined based on the capacity of cells to accumulate LC3B dots upon CQ treatment in cells from experiments described in (A), in function of their MT acetylation status. Autophagy flux was estimated by determining the average LC3B fluorescence intensity in cells (designated as ROIs). Then, cells with a mean LC3B fluorescence intensity above that average were considered as having high autophagy flux and cells with a mean LC3B fluorescence intensity below that value were considered as having low autophagy flux. A total of 331 cells were analyzed from n = 2 independent experiments.
