1. Introduction
2. Materials and Methods
2.1. Cell Culture and Chemical Treatments
2.2. Western Blotting Analysis
2.3. Immunofluorescence and Confocal Microscopy
2.4. Transfection of DNA or siRNA
2.5. Flow Cytometry for Determination of Autophagic Flux
2.6. Quantitative Polymerase Chain Reaction (qPCR)
2.7. Measurement of Lysosomal Aacidity and Lysosomal Enzyme Activity
2.8. Statistical Analysis
3. Results
3.1. Autophagic Flux Impairment in Human Fibroblasts Under Glucose Deprivation
3.2. Lysosomes Accumulate but Many Are Nonfunctional in Glucose-Deprived Cells
3.3. Lysosomes in Glucose-deprived Cells Exhibit Poor Acidity
3.4. Increased Reactive Oxygen Species Level Is also Involved in the Failure of Lysosome Acidification and Flux Blockade
3.5. Autophagic Flux Impairment Is Partially Relieved by Inhibition of ATM
3.6. Erk Activation Is Involved in the Impairment of Lysosome Acidity
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Name | Manufacturer | Cat # | dose used | Expected effect/property |
---|---|---|---|---|
chloroquine | Sigma-Aldrich, St. Louis, MO, USA | C6628 | 50 μM | Inhibition of autophagosome-lysosome fusion [35] |
bafilomycin A1 | Enzo Life Science, Farmingdale, NY, USA | BML-CM110-0100 | 200 nM | Inhibition of autophagosome-lysosome fusion and lysosome acidification [26] |
torin1 | Biorbyt, Cambridge, UK | orb146133 | 250 nM | Induction of autophagy by inhibiting mTOR [36] |
concanamycin A | Sigma-Aldrich | C9705 | 200 nM | Inhibition of vATPase [37] |
KU60019 | Selleckchem, Houston, TX, USA | S1570 | 0.5 μM | Inhibition of ATM [30] |
n-acetylcysteine | Sigma-Aldrich | A7250 | 5 mM | Antioxidant [38] |
4-hydroxy-2,2,6,6-tetramethyl-piperidin-1-oxyl | Sigma-Aldrich | 176141 | 1 mM | A membrane-permeable free radical scavenger [39] |
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