Figure 1.

Creation of a stable cell line serving as a reporter for autophagic flux.A, illustration depicting the use of optical pulse labeling (OPL) to measure autophagic flux. 1, Dendra2-LC3 is a photoconvertible fusion protein that irreversibly shifts its fluorescence from green to red upon exposure to 405 nm light. 2, Dendra2-LC3 is an autophagy substrate that is incorporated into autophagosomes. Prior to degradation, red fluorescence is high. 3, autophagosomes mature into autophagolysosomes, where photoconverted Dendra2-LC3 is degraded over time, resulting in a drop in red fluorescence intensity. The time-dependent decay of red signal serves as an estimate of autophagic flux, independent of new (green) LC3-Dendra2 synthesis. B, schematic for tagging native LC3 using CRISPR/Cas9 genome editing. In HEK293T cells, the Dendra2 ORF was introduced into the MAP1LC3B locus upstream of exon 1 creating an N-terminal fusion protein upon translation. C, western blot confirming the successful labeling of LC3 with Dendra2. Dendra2-LC3 HEK293T cells were treated with 20 nM siRNA targeting LC3 or scrambled siRNA. Lysates were collected after 48h and immunoblotted with an LC3 antibody, demonstrating the Dendra2-LC3 fusion protein running at the expected MW of 43 kDa that disappears upon siRNA-mediated knockdown of LC3. GAPDH serves as a loading control. D, Dendra2-LC3 reporter line imaged in the GFP and bright-field channels 48h after application of siRNA. Scale bar = 100 μm. E, Dendra2-LC3 cells imaged 6h after treatment with vehicle, 1 μM Torin1, and 20 nM Bafilomycin-A1. Scale bar = 10 μm.