Generation of an autophagic flux reporter sensitive to differential autophagy-inducing stressors. (A) Schematic representation of the autophagic flux reporter DsRed and GFP-tagged p62. p62 is selectively incorporated into and degraded along with the autophagosomal membrane. Therefore, the level of GFP-p62, relative to DsRed, is inversely proportional to autophagic flux. Examples of autophagy activation and inhibition were demonstrated using the histogram in the right panel. (B) HEK293A reporter cell line was treated with starvation in time- and concentration-dependent manners. WB was used to examine DsRed and GFP-p62 signals. FACS was employed to investigate GFP and DsRed fluorescence of the reporter cells treated with amino acid–free media (-AA) for 3 h. Histograms were used to depict changes in GFP-p62 and DsRed levels. (C) Reporter cell line was incubated with tunicamycin in time- and concentration-dependent manners. DsRed and GFP-p62 were analyzed using WB. FACS was used to examine GFP and DsRed signals of the reporter cells exposed to tunicamycin (10 µg/ml) for 6 h. Histograms were used to depict changes in GFP-p62 and DsRed levels. (D) Reporter cells were incubated with clofibrate in time- and concentration-dependent manners. DsRed and p62 levels were examined using WB. FACS was employed to investigate GFP and DsRed fluorescence of the reporter cells treated with clofibrate (1 mM) for 6 h. Histograms were used to depict changes in GFP-p62 and DsRed levels. Source data are available for this figure: SourceData F1.