Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Abstract

[Abstract unavailable

Increased transport of acetyl‐CoA into the endoplasmic reticulum causes a progeria‐like phenotype

“…Thus, the above results are not surprising. However, what remains to be determined is how the acetylation of Atg9a on K359 and K363 within the lumen of the ER can activate the core of the autophagy machinery, which is mainly cytosolic (Klionsky et al, 2016). Interestingly, the above two lysine residues are flanked by two coiled regions that could be involved with protein–protein interactions (Pehar et al, 2012).…”

Defective recruitment of motor proteins to autophagic compartments contributes to autophagic failure in aging

“…Autophagic flux was measured by immunoblot for LC3‐II and p62 (Klionsky, 2016) or by imaging with tandem reporter mCherry‐GFP‐LC3 (for in bulk autophagy) or mt‐Keima (Katayama et al., 2011; for mitophagy). Inhibition of lysosomal proteolysis for the flux assays was attained using 20 m m NH 4 Cl and 100 μ m leupeptin that we determined were saturating concentrations for blockage of LC3 degradation both in young and in old cells.…”

Role of the MAPK/cJun NH 2 -terminal kinase signaling pathway in starvation-induced autophagy

“…This observation suggested that MAPK8/9 deficiency caused no major change in autophagy as a result of MTOR inhibition. This conclusion was confirmed by measurement of autophagic flux by monitoring the formation of phosphatidylethanolamine-conjugated LC3B-II in response to MTOR inhibition in the presence of a lysosomal inhibitor [36] (Figure. 1(c) and S1B) and reduced accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1) (Figure.…”

“…Control studies demonstrated that rapamycin inhibited phosphorylation of the TORC1 substrate RPS6KB1 at Thr389, but not the TORC2 substrate AKT (thymoma viral proto-oncogene) at target site Ser473 (Figure 1(e)). We found that treatment of WT and mapk8 −/- mapk9 −/- immortalized MEFs with rapamycin caused a similar increase in autophagic flux that was measured by monitoring the accumulation of LC3B-II in the presence of a lysosomal inhibitor [36] (Figure. 1(f) and S1D).…”

“…Autophagy was also examined in cells treated with pharmacological inhibitors of MTOR (250 nM torin 1; Tocris, 4247) and TORC1 (200 nM rapamycin; Millipore Sigma, R8781) or under hypoxia (1.5% O 2 ) conditions. Autophagic flux was examined by measuring the LC3B-II:GAPDH ratio or LC3B-II:TUBA (tubulin alpha) ratio by immunoblot analysis, normalization of the data to the control condition (without autophagy induction or CQ), and calculation of the increased LC3B-II:GAPDH (or TUBA) ratio caused by treatment of the cells with 25 µM chloroquine diphosphate (CQ; Millipore Sigma, 25,745) to inhibit lysosomal protein degradation [36]. …”