Figure 3.

Effect of disease‐related mutant tau proteins on CMA. (a,b) Proteolysis of long‐lived proteins in mouse neuroblastoma cell line Neuro‐2a (N2a) treated with doxycycline to activate expression of the indicated tau proteins. Protein degradation was measured at the indicated times, in cells maintained in the presence (a) or absence (b) of serum, as the amount of acid‐precipitable radioactivity (amino acids and small peptides) released into the media. n = 3 different experiments with triplicate wells. (c–g) Same N2a cells as in a, were treated with doxycycline for 72 h and then transduced with lentivirus carrying the KFERQ‐PS‐Dendra2 reporter and maintained in the presence (c) or absence of serum (d) or in the presence of paraquat (PQ) (f) or thapsigargin (TG) (g) at the indicated concentrations. Representative images of cells maintained in the presence (c) or absence (d) of serum 16 h after photoswitching. Insets show higher magnification. Nuclei were stained with DAPI. (e) Quantification of average number of puncta per cell section in experiments as the ones in c and d. (f–g) Quantification of average number of puncta per cell in absolute values (left) or relative to the number in untreated cells (right) in experiments as in c but in the presence of paraquat (f) or thapsigargin (g). n > 800 cells/condition in three different experiments with triplicate wells. All values are mean ± SEM. Differences are significant for *P < 0.05 and **P < 0.01.