title: Dynamic imaging of autophagy-lysosomal pathway and autophagy function following pulmonary hypoxia/reoxygenation in vitro.
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.
authors: Department of Thoracic Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
authors: Tian-Shu
authors: TS
authors: Liu
authors: Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
authors: Yi-Ting
authors: YT
authors: Cai
authors: Department of Thoracic Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
authors: Zhi-Fu
authors: ZF
authors: Mao
authors: Department of Thoracic Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
authors: Jie
authors: J
authors: Huang
authors: Department of Thoracic Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
authors: Tao
authors: T
authors: Fan
authors: Department of Thoracic Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, China. 936886731@qq.com.
authors: Qing
authors: Q
authors: Geng
mesh_terms: D000818:Animals; D001343:Autophagy; D001483:Base Sequence; D017931:DNA Primers; D000860:Hypoxia / Q000503:physiopathology*; D066298:In Vitro Techniques; D008168:Lung / Q000503:physiopathology*; D008247:Lysosomes / Q000502:physiology*; D010102:Oxygen Inhalation Therapy; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D017931:DNA Primers