Dynamic imaging of autophagy-lysosomal pathway and autophagy function following pulmonary hypoxia/reoxygenation in vitro.
10.1007/s11596-015-1428-z
- Author:
Tian-shu LIU
1
;
Yi-ting CAI
;
Zhi-fu MAO
;
Jie HUANG
;
Tao FAN
;
Qing GENG
Author Information
1. Department of Thoracic Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, China, 459673598@qq.com.
- Publication Type:Journal Article
- MeSH:
Animals;
Autophagy;
Base Sequence;
DNA Primers;
Hypoxia;
physiopathology;
In Vitro Techniques;
Lung;
physiopathology;
Lysosomes;
physiology;
Oxygen Inhalation Therapy;
Rats;
Real-Time Polymerase Chain Reaction
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2015;35(2):302-308
- CountryChina
- Language:English
-
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.
