Figure 2.

Time-dependent decay of Dendra2-LC3 serves as an accurate measure of autophagic flux.A, Dendra2-LC3 HEK293T cells were imaged prior to photoconversion to measure background RFP intensity. Immediately following photoconversion, cells were treated with DMSO, 1 μM Torin1, or 10 nM Bafilomycin-A1 and imaged at the indicated times. Images are pseudocolored to better highlight intensity differences. Scale bar = 50 μm. B–E, time-dependent changes in photoconverted Dendra2-LC3 fluorescence in the RFP (B and C) and GFP (D and E) channels. Intensity measurements were obtained prior to (dark gray) and following photoconversion (light gray) and normalized. For RFP measurements, the background intensity prior to conversion was set to 0 and the postconversion value to 1. GFP values are scaled to the preconversion intensity. Error bars represent SEM from three replicate experiments. B, treatment with 1 μM Torin1 accelerates Dendra2-LC3 decay, reflecting enhanced autophagic degradation of the reporter, while treatment with bafilomycin-A1 stabilizes reporter half-life. D, photoconversion results in a 40% drop in GFP intensity. As new Dendra2-LC3 is synthesized, GFP levels return to prephotoconversion levels over 13.5 h. Torin1 blocks the observed return in GFP fluorescence by accelerating flux. Genetic inhibition of autophagy via siRNA-mediated knockdown of ATG5 2 days prior attenuates Torin1’s effects in both the RFP (C) and GFP (E) channels. For (B–D), Two-Way ANOVA was performed, indicating significant effects of both time and treatment, as well as a significant interaction between time and treatment. These values are summarized in Table S1. ∗ denotes p < 0.05 using DMSO as reference group with Tukey’s multiple comparisons test; # indicates p < 0.05 with the scramble control for each drug treatment as the reference group (i.e., scramble siRNA 1 μM Torin1 versus ATG5 siRNA 1 μM Torin1). Superscript number indicates the first time point when significance was achieved.