Mrfp gfp lc3 adenovirus construct

Cells at 70–80% confluence were transfected with pcDNA3.1-GFP-LC3 with Lipo2000. The cells were then observed under fluorescence microscope and the cells with at least 3 green dots were considered autophagic cells. The percentage of autophagy = autophagic cells / total cells x 100%. For observation under electron microscope, cells washed with PBS were pelleted and the cell pellet was fixed in 2.5% glutaric dialdehyde and 1% osmic acid for 2 hr. After the wash, the pellet was dehydrated and embedded in epoxy resin. The ultra-thin sections (45 nm) were stained with lead acetate, and the stained sections were observed under JEM-2000EX electron microscope [16 (link)]. To monitor autophagic flux, the tandem mRFP-GFP-LC3 adenovirus construct obtained from Hanbio Inc (Shanghai, China) was used in this study. The exhibited green fluorescent protein (GFP) and red fluorescent protein (RRP) relied on different sensitivity of pH to monitor progression from the autophagic flux. In brief, mRFP-GFP-LC3 construct capitalized on the pH difference between the acidic autolysosome and the neutral autophagosome to monitor progression from the autophagosome to autolysosome by using confocal microscopy [17 ].

mRFP-GFP-LC3 adenovirus transfection was used to monitor autophagic flux by marking and tracking LC3. The mRFP–GFP–LC3 adenovirus construct was obtained from Hanbio Inc. HUVECs were transfected with mRFP–GFP–LC3 adenovirus for 24 h following the manufacturer’s instructions and then transfected with siRNA or plasmid for 48 h. The cells were then transferred to a confocal dish and treated with H2O2 after the cells adhered. Finally, a confocal microscope was used to observe the cells and acquire images (Nikon A1R, Tokyo, Japan).