Fig 2: The Proteasome Is a Selective Target of Autophagy(A) Frequency of selected UNIPROT annotation terms in the basal (blue bars, n = 343) and LPS-associated (red bars, n = 241) autophagy substrate proteome. The annotation for proteasomes is marked by a green arrow.(B) Stacked line graphs depicting the time structure of proteasome subunit turnover via autophagy (n = 24). Top graph, basal livers. Bottom graph, LPS-treated livers. As a visual aid, turnover profiles for 11S regulatory subunits are black, those for 19S subunits are green and brown, and those for 20S subunits are blue and red.(C) Enrichment analysis for the UNIPROT annotation terms depicted in (A). Bars pointed to the right denote over-enrichment, and bars pointed to the left denote under-enrichment. See Data S4 for a tabular presentation of these data. *p < 0.05 (χ2 contingency table analysis).(D) Western blot depicting subcellular fractionation of total liver protein using Nycodenz density gradient centrifugation (see STAR Methods). LAMP2 and LDH are provided as lysosomal and cytosolic markers, respectively. Higher fraction numbers denote increasing density. Left, sham (PBS)-treated mice. Right, leupeptin-treated mice. High-density fractions containing autophagic substrates are denoted by arrows and yellow highlights. Data are representative of 3 independent analyses.(E) Western blots of mouse liver ER separated by OptiPrep density ultracentrifugation (see STAR Methods). Peak fractions for ATG9a and LC3b-II are highlighted yellow. Sec61 is provided as an ER marker. Data are representative of 3 independent analyses.(F) Mean proteasome enrichment relative to ribosomes ± SE (n = 3), as reflected by PSMB2/RS3 ratios. Peak fractions for ATG9a and LC3b-II are denoted by a gray bar.

Fig 3: Upregulated autophagic activity in the TBI and PF groups, but not in TBI + PF. (A) The autophagy-related proteins p62 and LC3B II can be used as reporters to measure autophagic activity by immunoblotting. Usage of the autophagy inhibitor Baf A1 allows the evaluation of autophagic flux. Experiments were performed on cell lysates of three independent cell cultures and representative blots are shown. (B) LC3B I, LC3B II and p62 bands detected on the immunoblots were quantified from three independent experiments and the obtained densitometric values are displayed as mean ± SEM. (C) Static observations of the cellular levels of LC3B and p62 cannot be used to draw conclusions about the actual lysosomal turn-over. However, when comparing the results in the absence (−) and presence of the inhibitor Baf A1 we determined an increase in autophagic flux. (D) Schematic representation of the data shown in (C). Mean values are presented and were used to illustrate how autophagosome synthesis and degradation can be calculated. (E) The amount of autophagosomes that would have been degraded shows a tendency for an increase in the TBI and PF groups, but not in TBI + PF. (F) Correspondingly, autophagosome synthesis calculations showed the same trend, though also not statistically significant. Data in (C), (E) and (F) are displayed as mean with SD and were obtained from three independent cell cultures. Statistical analysis was performed using one-way ANOVA (B,E,F) and two-way ANOVA (C) followed by Tukey’s multiple comparison tests, *p ≤ 0.05 **p ≤ 0.01 ***p ≤ 0.001.

Fig 4: Autophagy markers abnormally accumulate in SCA2 patients’ brain.Immunohistochemical analysis of cerebellum post-mortem brain sections of SCA2 patients and healthy controls. A Staining for ataxin-2 showed no immunoreactivity in cerebellum tissue of healthy control. D On the other hand, staining for ataxin-2 revealed the presence of aggregated structures in cerebellum of SCA2 brain, indicating a disease state. Staining for LC3B and SQSTM1 autophagic markers demonstrated abnormal puncta accumulation of these proteins in SCA2 cerebellum (E, F), compared to healthy individual tissues (B, C).

Fig 5: Epithelial injury promotes accumulation of p62Paraffin-embedded sections prepared from the intestinal or colonic tumors isolated from untreated (Un), DSS-treated or AOM+DSS-treated Apc1638N/+ (A) or CR-infected ApcMin/+ (B) mice were co-stained with antibodies for p62 and Dclk1 (A, B), Dclk1 and β-catenin or Dclk1 and EZH2, respectively (B). Inset in B shows merged images of the p62/Dclk1 puncta. Scale bars = 150-250μm; n = 3 independent experiments. (C) Electron microscopy showing presence of autophagic vesicles in the CR-infected but not uninfected ApcMin/+ mouse distal colonic fragments (2μm sections; arrows; n = 3 independent experiments). (D) Western blotting to detect changes in Dclk1, p62, LC3B and EZH2, respectively. GAPDH and β-Actin were used as loading controls. (E) Evidence of p62 and Dclk1 co-localization in surgically resected colon cancer samples with metastasis to the liver. Paraffin-embedded sections prepared from surgically resected colon tumors and liver mets were co-stained with antibodies for p62 and Dclk1 and p62 and LC3B, respectively. Representative merged images are shown with an H&E stained section showing the gross morphology. Scale bars = 450μm.