Protocol detail

Doxorubicin and Autophagy Modulation

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Doxorubicin was from Selleckchem (Houston, TX). HPβCD was from Cayman Chemical Company (Ann Arbor, MI). 10-(4′-(N-diethylamino)butyl)-2-chloro-phenoxazine hydrochloride (10-NCP) was from Calbiochem (San Diego, CA). NH4Cl was from Biomatik (Wilmington, DE). Lysotracker Green DND-26 was from Life Technologies (Carlsbad, CA). Antibodies against actin (#3700, 1:1.000) and p62 (#5114, 1:1.000) were from Cell Signaling (Danvers, MA). Antibody against tubulin (#ab7751, 1:1.000) was from Abcam (Cambridge, MA). Antibody against MAP2c (#sc-74421, 1:100) was from Santa Cruz Biotechnologies (Dallas, TX), and antibody against LC3 (#PD014, 1:1.000) was from MBL (Woburn, MA). Anti-mouse Alexa Fluor 488-labeled (#A11001, 1:200) was from Life Technologies. Hoechst dye was from Santa Cruz Biotechnology. pEGFP-N1-TFEB was from Addgene (#38119, deposited by Dr. Shawn Ferguson, Yale School of Medicine). pGW1-beclin1-GFP was described [37 (link)]. pGW1-GFP-p62 was cloned from pMXs-puro GFP-p62, which was from Addgene (#38277, deposited by Dr. Noboru Mizushima, the University of Tokyo).

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Moruno Manchon J.F., Uzor N.E., Kesler S.R., Wefel J.S., Townley D.M., Nagaraja A.S., Pradeep S., Mangala L.S., Sood A.K, & Tsvetkov A.S. (2016). TFEB ameliorates the impairment of the autophagy-lysosome pathway in neurons induced by doxorubicin. Aging (Albany NY), 8(12), 3507-3519.

Publication 2016

Doxorubicin Lysotracker Green DND-26 Anti-mouse Alexa Fluor 488-labeled Hoechst dye pEGFP-N1-TFEB pMXs-puro GFP-p62

Actin Alexa fluor 488 Antibodies Antibody Beclin1 Cayman Doxorubicin Lysotracker Mouse Phenoxazine Tfeb Tubulin

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Variable analysis

independent variables

Doxorubicin
HPβCD
10-(4′-(N-diethylamino)butyl)-2-chloro-phenoxazine hydrochloride (10-NCP)
NH4Cl
PEGFP-N1-TFEB
PGW1-beclin1-GFP
PGW1-GFP-p62

dependent variables

Lysotracker Green DND-26
Actin
Tubulin
MAP2c

control variables

Hoechst dye

positive controls

Not specified

negative controls

Not specified

Annotations

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