Dynamic imaging of autophagy-lysosomal pathway and autophagy function following pulmonary hypoxia/reoxygenation in vitro / 华中科技大学学报(医学)(英德文版)
Journal of Huazhong University of Science and Technology (Medical Sciences)
;
(6): 302-308, 2015.
Article
in English
| WPRIM
| ID: wpr-331069
ABSTRACT
Alterations of the autophagy-lysosomal pathway (ALP) and autophagy have been involved in lung ischemia-reperfusion (I/R) injury. However, dynamic imaging of ALP function under lung I/R injury particularly is not fully understood. Here we depicted the live-cell fluorescence imaging of autophagosome to monitor ALP activation and autophagy function. The pAsRed2-N1-LC3 vectors were transfected into CRL-2192 NR8383 (an alveolar macrophage cell line) and CCL149 (an alveolar epithelial cell line) successfully. 0-h, 2-h, 4-h, and 6-h hypoxia/0-h, 2-h, 4-h, and 6-h reoxygenation were then induced with an ALP inhibitor (3-MA) or activator (rapamycin) in the culture of transfected cells separately. ALP activation was conformed by up-regulating AMPK and beclin1 expression. Apoptosis was not obvious in 2-h hypoxia/2-h reoxygenation. pAsRed2-N1-LC3 CCL149 and pAsRed2-N1-LC3 NR8383 cells revealed gradually enhanced AsRed2 from 2-h to 6-h hypoxia/reoxygenation. AsRed2 varied sensitively to 3-MA and rapamycin interventions during 2-h hypoxia/reoxygenation. Our data provides a simple method of autophagosome imaging to monitor ALP activation and autophagy function in lung I/R injury.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Oxygen Inhalation Therapy
/
Physiology
/
Autophagy
/
In Vitro Techniques
/
Base Sequence
/
DNA Primers
/
Real-Time Polymerase Chain Reaction
/
Lung
/
Lysosomes
/
Hypoxia
Limits:
Animals
Language:
English
Journal:
Journal of Huazhong University of Science and Technology (Medical Sciences)
Year:
2015
Type:
Article
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