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In Situ Detection of Starvation-induced Autophagy

Wim Martinet, Guido R.Y. De Meyer, Luc Andries, Arnold G. Herman and Mark M. Kockx

Division of Pharmacology, University of Antwerp, Wilrijk, Belgium (WM,GRYDM,AGH,MMK); HistoGeneX, Edegem, Belgium (LA); and Department of Pathology, General Hospital Middelheim, Antwerp, Belgium (MMK)

Correspondence to: Dr. Wim Martinet, Division of Pharmacology, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium. E-mail: wim.martinet{at}ua.ac.be

Autophagy is a regulated bulk degradation process involved in many different human pathologies. Transmission electron microscopy (TEM) is currently the only reliable method for monitoring autophagy in situ. Because TEM is labor intensive, we questioned whether useful marker proteins can be found for unambiguous detection of autophagy in tissue via routinely used colorimetric, immunohistochemical, or fluorescent techniques. Starved HepG2 hepatocytes and nutrient deprived liver tissue were used as a model for the initiation of autophagy. Our findings indicate that starvation-induced autophagy in HepG2 cells was associated neither with differential mRNA gene expression nor with changes in the expression level of known autophagy-related proteins. On the contrary, both transcription and translation were inhibited, suggesting that the identification of autophagy-specific biomarkers for tissue is highly compromised. Light chain 3 (LC3) protein, which is an attractive marker of autophagosomes, revealed a relatively low expression level in tissue and cultured cells, but could be detected via immunohistochemistry in liver from GFP-LC3 transgenic mice. The number of LC3 immunopositive dot-like structures was significantly upregulated in liver tissue from nutrient-deprived GFP-LC3 mice as compared with nonstarved control tissue. Our results suggest that LC3 immunostaining can be used as an alternative detection method for autophagy in situ, but only when this protein is overexpressed. (J Histochem Cytochem 54:85–96, 2006)

Key Words: amino acid deprivation • autophagy • cell death • liver • HepG2 cells • gene expression • LC3

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