Fig 2: Acetylated tau has an inhibitory effect on CMA activity.a Representative images of mouse neuroblastoma cell line Neuro-2a (N2a) transduced with KFERQ-PS-Dendra2 and transfected with empty vector (Control), FLAG-wild-type tau (WT Tau) or FLAG-K274,281Q tau (KQ Tau), and maintained in the presence (+) or absence (-) of serum for 16 h. Nuclei are highlighted with DAPI in blue. Insets show higher magnification images and arrows point examples of fluorescent puncta. n > 800 cells/condition in three different wells and 4 independent experiments. b Quantification of an average number of puncta per cell section. [Two-way ANOVA Tau status effect: F(2,12) = 179.1, p < 0.0001], n > 800 cells/condition in three different wells, and n = 4 independent experiments. Controls for tau expression are shown in Supplemental Fig. 1h (protein levels) and Supplemental Fig. 3a (mRNA levels). c Degradation rates of long-lived proteins in N2a cells transfected as in a and radiolabeled for 48 h with 3H-leucine), [One-way ANOVA Tau status effect: F(2,6) = 4;619, p = 0.0610]. Right: fraction of degradation occurring in lysosomes (sensitive to inhibition of lysosomal proteolysis), [One-way ANOVA Tau status effect: F(2,6) = 14.55, p = 0.005], n = 3 independent experiments. d Immunoblot for L2A of isolated lysosomes subjected to blue-native electrophoresis after being incubated alone (No protein) or with unmodified (Tau) or in vitro acetylated tau (Ac-Tau). A 700-kDa multimeric complex and the 110-kDa monomer are shown. Right: Quantification of multimeric levels of L2A relative to those in lysosomes incubated alone. [One-way ANOVA Tau status effect: F(2,13) = 5.580, p = 0.0178], n = 4 independent experiments. e Lysosomes incubated and processed under the same conditions as in d but in the presence or absence of hsc70. f Immunoblot for tau of the GST-pulldowns of GST-hsc70 incubated with tau or Ac-Tau at neutral pH for 30 min at 37 °C. Input (Ipt): 1/10 of tau added to the incubation. Right: Quantification of tau bound to Hsc70. [Two-tailed t-test t4 = 4.432, p = 0.0114], n = 3. g Samples incubated as in f but at different pH. Right: Quantification of tau bound to hsc70 [Two-way ANOVA Tau status effect: F(1,18) = 10.85, p = 0.0040], n = 4 independent experiments. h Tau or K280Q tau binding assay to immobilized hsc70 determined by ELISA at the indicated pH. See also Supplemental Fig. 3b and c for binding assays at intermediate pH. n = 6 (in 2 independent experiments). i Summary plot of the binding assay at all tested pH for tau and acetylated tau (Ac-Tau). [Two-way ANOVA Tau status effect: F(1,8) = 42.10, p = 0.0002]. n = 6 (in 2 independent experiments). j Immunoblots of lysosomes from starved rat livers, pretreated or not with protease inhibitors (PI) and/or NH4Cl 20 mM for 10 min at 4 °C and then incubated with tau (left) or Ac-Tau (middle). n = 3 independent experiments. k Quantification of tau proteins binding and uptake by lysosomes. [Two-way ANOVA Tau status effect: F(1,17) = 17.22, p = 0.0013], n = 3 independent experiments. All values are mean ± s.e.m. Differences with control or unmodified tau were significant for *p < 0.05, **p < 0.01, ***p < 0.005. Uncropped blots are shown in Supplemental Fig. 7.

Fig 3: Lysosomal isolation and mass spectrometry analysis of constitutive resident proteins. (a) Western blot analysis for LAMP1, LAMP2A, CTSD, GBA, and HSC70 in homogenate (Total), cytosol (Cyt), and lysosomes (Lys) with high CMA activity (CMA) or high MA activity (MA) isolated from control or palbociclib-treated SK-MEL-103 cells. Arrows indicate precursor and mature form. Ponceau staining is shown as loading control. (b) Scheme of the hypothetical changes in levels of proteins in lysosomes treated or not with ammonium chloride and leupeptin (N/L) 16 h before isolation. Proteins are classified depending on these changes as degraded (lysosomal substrates) if they accumulate upon N/L treatment, or constitutive (resident proteins) if they do not accumulate upon N/L treatment. (c) Principal component analysis (PCA) of the CMA and MA lysosome samples, n = 3 with two technical replicates each. (d) Changes in abundance of the lysosomal constitutive proteins in CMA and MA lysosomes. Constitutive proteins were defined as those that do not change significantly p > 0.05 in N/L versus vehicle or that have a negative log2 fold change N/L versus vehicle. (e) Top lysosomal resident proteins found at higher or lower levels in CMA and MA lysosomes. (f) Fold change (over control) in protein levels of TOR protein components found in both CMA and MA lysosomes.

Fig 4: Effect of long-term treatment with D-cysteine on the amounts of Nrf2- and CMA-related proteins in cerebellar lysates from ICR mice. (A) Immunoblot analyses of Nrf2, NQO1, LAMP2A, Hsc70, MEF2D, and ß-actin in cerebellar lysates from ICR mice daily treated with saline (Sal) and D-cysteine (100 mg/kg/day) for 10 weeks. Whole blot images are presented in Figure S6. (B) Quantitative analyses of the amounts of Nrf2, NQO1, LAMP2A, Hsc70, and MEF2D shown in A. Amounts of ß-actin were used as internal controls for the quantification. * p < 0.05, ** p < 0.01 vs. saline-treated mice (unpaired t-test, n = 5 in both saline- and D-cysteine-treated mice).

Fig 5: The direct association between NCoR1 and Hsc70. Primary hepatocytes were isolated from 8- to 10-week-old male C57BL/6 mice and treated with siRNA or chemicals as indicated. (A) Co-immunoprecipitation of endogenous Hsc70 and NCoR1 was performed with IgG or Hsc70 antibody in primary hepatocytes. (B) Mouse primary hepatocytes expressing LAMP2A-mCherry (red) were incubated with leupeptin 100 µM for 24 h. The fixed cells were subjected to immunofluorescence analysis with antibodies against NCoR1 (green). Nuclei were stained with DAPI (blue). Representative confocal microscopy images are shown, and the scale bar is set to 10 µm. The percentage of LAMP2A-mCherry spots colocalizing with NCoR1 was plotted in left panel. Each bar represents the mean ± SD. Asterisks above the bars indicate significant differences compared to the control using Student's t-test: ***p < 0.001.