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mCherry-GFP-LC3 reporter assay

WW Wenjun Wang
JL Jianshuang Li
JT Junyang Tan
MW Miaomiao Wang
JY Jing Yang
ZZ Zhi-Min Zhang
CL Chuanzhou Li
AB Alexei G. Basnakian
HT Hong-Wen Tang
NP Norbert Perrimon
QZ Qinghua Zhou
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mCherry-GFP-LC3 reporter plasmid which expressed LC3 fused with mCherry and GFP, were used to detect the autophagic flux. The green fluorescent signal of GFP is sensitive to the acidic environment of lysosomes, whereas the red fluorescent signal of mCherry is more stable. Therefore, colocalization of GFP and mCherry fluorescence (yellow puncta) indicated an autophagosome, and detection of mCherry but not GFP (red puncta) indicated an autolysosome. Cells were transfected with the mCherry-GFP-LC3 reporter plasmids for 48 h. After the treatment, cells were fixed and imaged. Yellow and red puncta were counted for each cell line. At least 100 cells were analyzed per treatment condition.

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