Cells where treated with Bafilo alone without any stresser or autophagy inducer
Dear Modar,
The following paper covers the answer to your question:
PLoS One. 2014; 9(4): e93257.
Published online 2014 Apr 2. doi: 10.1371/journal.pone.0093257
PMCID: PMC3973706
ATP6V0C Knockdown in Neuroblastoma Cells Alters Autophagy-Lysosome Pathway Function and Metabolism of Proteins that Accumulate in Neurodegenerative Disease
Leandra R. Mangieri,#1 Burton J. Mader,#1,3 Cailin E. Thomas,1 Charles A. Taylor,1 Austin M. Luker,1 Tonia E. Tse,1Carrie Huisingh,2 and John J. Shacka1,2,*
Srinivasa M. Srinivasula, Editor
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Abstract
ATP6V0C is the bafilomycin A1-binding subunit of vacuolar ATPase, an enzyme complex that critically regulates vesicular acidification. We and others have shown previously that bafilomycin A1 regulates cell viability, autophagic flux and metabolism of proteins that accumulate in neurodegenerative disease. To determine the importance of ATP6V0C for autophagy-lysosome pathway function, SH-SY5Y human neuroblastoma cells differentiated to a neuronal phenotype were nucleofected with non-target or ATP6V0C siRNA and following recovery were treated with either vehicle or bafilomycin A1 (0.3–100 nM) for 48 h. ATP6V0C knockdown was validated by quantitative RT-PCR and by a significant decrease in Lysostracker Red staining. ATP6V0C knockdown significantly increased basal levels of microtubule-associated protein light chain 3-II (LC3-II), α-synuclein high molecular weight species and APP C-terminal fragments, and inhibited autophagic flux. Enhanced LC3 and LAMP-1 co-localization following knockdown suggests that autophagic flux was inhibited in part due to lysosomal degradation and not by a block in vesicular fusion. Knockdown of ATP6V0C also sensitized cells to the accumulation of autophagy substrates and a reduction in neurite length following treatment with 1 nM bafilomycin A1, a concentration that did not produce such alterations in non-target control cells. Reduced neurite length and the percentage of propidium iodide-positive dead cells were also significantly greater following treatment with 3 nM bafilomycin A1. Together these results indicate a role for ATP6V0C in maintaining constitutive and stress-induced ALP function, in particular the metabolism of substrates that accumulate in age-related neurodegenerative disease and may contribute to disease pathogenesis.
Hoping this will be helpful,
Rafik
Bafilo increase pH in lysosome and block the completion of the final step of autophagy.
Hi Modar,
Using bafilomycin will inhibit the basal level of autophagy when used at low concentrations (200nM) for short periods of time (unto 6h) after that it also starts affecting eadosomal processes, due to its effect on the acidification of lysosomes. This is commonly used to assess the basal rate of autophagy under any given circumstance (e.g.. new cell line, depletion of gene X, drug treatment etc).
Also the guidelines for assessing autophagy flux (link below) can help with interpreting the results. all the best
david
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404883/pdf/auto-8-445.pdf
Dear Modar,
Cells treated with Bafilomycin A1(100nM) increase LC3II which indicates prevention of maturation of autophagosome at the end stage or before fusion with the lysosome.LC3II will not be degraded and appears on the surface of autophagy vacuole.
regards
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