AUTOCOUNTER, an ImageJ JavaScript to Analyze LC3B-GFP Expression Dynamics in Autophagy-induced Astrocytoma Cells

Overview

Journal Eur J Histochem

Specialty Biochemistry

Date 2013 Jan 31

PMID 23361240

Citations 14

Authors

L Fassina

G Magenes

A Inzaghi

S Palumbo

G Allavena

C Miracco

L Pirtoli

M Biggiogera

S Comincini

Affiliations

Soon will be listed here.

Abstract

An ImageJ JavaScript, AUTOCOUNTER, was specifically developed to monitor and measure LC3B-GFP expression in living human astrocytoma cells, namely T98G and U373-MG. Discrete intracellular GFP fluorescent spots derived from transduction of a Baculovirus replication-defective vector (BacMam LC3B-GFP), followed by microscope examinations at different times. After viral transgene expression, autophagy was induced by Rapamycin administration and assayed in ph-p70S6K/p70S6K and LC3B immunoblotting expression as well as by electron microscopy examinations. A mutated transgene, defective in LC3B lipidation, was employed as a negative control to further exclude fluorescent dots derived from protein intracellular aggregation. The ImageJ JavaScript was then employed to evaluate and score the dynamics changes of the number and area of LC3B-GFP puncta per cell in time course assays and in complex microscope examinations. In conclusion, AUTOCOUNTER enabled to quantify LC3B-GFP expression and to monitor dynamics changes in number and shapes of autophagosomal-like vesicles: it might therefore represent a suitable algorithmic tool for in vitro autophagy modulation studies.

Citing Articles

PICALM Regulating the Generation of Amyloid β-Peptide to Promote Anthracycline-Induced Cardiotoxicity.

Bao M, Hua X, Chen X, An T, Mo H, Sun Z Adv Sci (Weinh). 2024; 11(32):e2401945.

PMID: 38935046 PMC: 11348153. DOI: 10.1002/advs.202401945.

An improved method for high-throughput quantification of autophagy in mammalian cells.

Koepke L, Winter B, Grenzner A, Regensburger K, Engelhart S, van der Merwe J Sci Rep. 2020; 10(1):12241.

PMID: 32699244 PMC: 7376206. DOI: 10.1038/s41598-020-68607-w.

Transient inhibition of mTOR in human pluripotent stem cells enables robust formation of mouse-human chimeric embryos.

Hu Z, Li H, Jiang H, Ren Y, Yu X, Qiu J Sci Adv. 2020; 6(20):eaaz0298.

PMID: 32426495 PMC: 7220352. DOI: 10.1126/sciadv.aaz0298.

Midkine signaling maintains the self-renewal and tumorigenic capacity of glioma initiating cells.

Lopez-Valero I, Davila D, Gonzalez-Martinez J, Salvador-Tormo N, Lorente M, Saiz-Ladera C Theranostics. 2020; 10(11):5120-5136.

PMID: 32308772 PMC: 7163450. DOI: 10.7150/thno.41450.

Ultrastructural histochemistry in biomedical research: Alive and kicking.

Malatesta M Eur J Histochem. 2018; 62(4).

PMID: 30418011 PMC: 6250102. DOI: 10.4081/ejh.2018.2990.

References

Jackson W, Giddings Jr T, Taylor M, Mulinyawe S, Rabinovitch M, Kopito R . Subversion of cellular autophagosomal machinery by RNA viruses. PLoS Biol. 2005; 3(5):e156. PMC: 1084330. DOI: 10.1371/journal.pbio.0030156. View

Bampton E, Goemans C, Niranjan D, Mizushima N, Tolkovsky A . The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes. Autophagy. 2006; 1(1):23-36. DOI: 10.4161/auto.1.1.1495. View

Kraft C, Deplazes A, Sohrmann M, Peter M . Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease. Nat Cell Biol. 2008; 10(5):602-10. DOI: 10.1038/ncb1723. View

Kaniuk N, Kiraly M, Bates H, Vranic M, Volchuk A, Brumell J . Ubiquitinated-protein aggregates form in pancreatic beta-cells during diabetes-induced oxidative stress and are regulated by autophagy. Diabetes. 2007; 56(4):930-9. DOI: 10.2337/db06-1160. View

Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E . A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res. 2001; 61(2):439-44. View

Klionsky D, Abeliovich H, Agostinis P, Agrawal D, Aliev G, Askew D . Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy. 2008; 4(2):151-75. PMC: 2654259. DOI: 10.4161/auto.5338. View

Klionsky D, Meijer A, Codogno P . Autophagy and p70S6 kinase. Autophagy. 2006; 1(1):59-60. DOI: 10.4161/auto.1.1.1536. View

Pankiv S, Clausen T, Lamark T, Brech A, Bruun J, Outzen H . p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem. 2007; 282(33):24131-45. DOI: 10.1074/jbc.M702824200. View

Mizushima N, Yoshimori T . How to interpret LC3 immunoblotting. Autophagy. 2007; 3(6):542-5. DOI: 10.4161/auto.4600. View

10.

ARSTILA A, Trump B . Autophagocytosis: origin of membrane and hydrolytic enzymes. Virchows Arch B Cell Pathol. 1969; 2(1):85-90. DOI: 10.1007/BF02889572. View

11.

Kogel D, Fulda S, Mittelbronn M . Therapeutic exploitation of apoptosis and autophagy for glioblastoma. Anticancer Agents Med Chem. 2010; 10(6):438-49. DOI: 10.2174/1871520611009060438. View

12.

Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T . LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000; 19(21):5720-8. PMC: 305793. DOI: 10.1093/emboj/19.21.5720. View

13.

Yang Z, Klionsky D . An overview of the molecular mechanism of autophagy. Curr Top Microbiol Immunol. 2009; 335:1-32. PMC: 2832191. DOI: 10.1007/978-3-642-00302-8_1. View

14.

Barbieri G, Palumbo S, Gabrusiewicz K, Azzalin A, Marchesi N, Spedito A . Silencing of cellular prion protein (PrPC) expression by DNA-antisense oligonucleotides induces autophagy-dependent cell death in glioma cells. Autophagy. 2011; 7(8):840-53. DOI: 10.4161/auto.7.8.15615. View

15.

Kamada Y, Funakoshi T, Shintani T, Nagano K, Ohsumi M, Ohsumi Y . Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J Cell Biol. 2000; 150(6):1507-13. PMC: 2150712. DOI: 10.1083/jcb.150.6.1507. View

16.

Mizushima N . Autophagy: process and function. Genes Dev. 2007; 21(22):2861-73. DOI: 10.1101/gad.1599207. View

17.

Hundeshagen P, Hamacher-Brady A, Eils R, Brady N . Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy. BMC Biol. 2011; 9:38. PMC: 3121655. DOI: 10.1186/1741-7007-9-38. View

18.

Szeto J, Kaniuk N, Canadien V, Nisman R, Mizushima N, Yoshimori T . ALIS are stress-induced protein storage compartments for substrates of the proteasome and autophagy. Autophagy. 2006; 2(3):189-99. DOI: 10.4161/auto.2731. View

19.

Munafo D, Colombo M . A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci. 2001; 114(Pt 20):3619-29. DOI: 10.1242/jcs.114.20.3619. View

20.

Niemann A, Baltes J, Elsasser H . Fluorescence properties and staining behavior of monodansylpentane, a structural homologue of the lysosomotropic agent monodansylcadaverine. J Histochem Cytochem. 2001; 49(2):177-85. DOI: 10.1177/002215540104900205. View