GFP-LC3 stable reporter cell line for detecting the rate of autophagy and for drug screening In (A), without Selective Permeabilization no shift of GFP-LC3 level is detected using flow cytometry before and after starvation (induction of autophagy). The position of the histograms indicates the high level of GFP-LC3 expression in the cytoplasm.
In (B), with Selective Permeabilization GFP-LC3 level remains high in autophagosomes when starved in the presence of lysosome inhibitor (green); even without the inhibitor, a slight shift is observed when starved (blue). All the cytosolic GFP-LC3 is washed away if no autophagy is induced by starvation (gray).

Image analysis of GFP-LC3 reporter cell line Cells are nutrient deprived to induce autophagy and then treated with autophagy reagent A to prolong the signal for detection of translocated GFP-LC3 in the autophagosomes, which is a hallmark of the autophagic process (B). As illustrated, GFP-LC3 puncta is visualized as green dots. Control cells which are uninduced are shown in (A). Cell nuclei stained with Dapi (blue).

Rapamycin induces Autophagy through the mTOR pathway Rapamycin is an inhibitor of the mTOR pathway, and by targeting mTOR, rapamycin mimics the cellular starvation response and leads to activation of autophagy as illustrated by the right shift of the histogram (green). Cells were treated with 400 nM Rapamycin for 48 hours prior to data acquisition.

Dynasore inhibits Autophagy by inhibition of autophagosome formation Dynasore is a cell-permeable inhibitor of dynamin. Dynamin is essential for clathrin-dependent coated vesicle formation. Dynasore acts as a potent inhibitor of endocytic pathways known to depend on dynamin by rapidly blocking coated vesicle formation within seconds of dynasore addition. As a result, Dynasore will inhibit autophagosome formation, which in effect, will inhibit autophagy as illustrated by the left shift of the histogram (green). Cells were treated with 80 uM Dynasore for 3 hours prior to data acquisition.

Selective Permeabilization helps discriminate cytosolic from autophagic LC3 Discrimination between GFP-cytosolic LC3-I from autophagosome associated GFP-LC3-II is achieved by disruption of cell PM by using an autophagy enabling solution (Autophagy Reagent B).
This selective permeabilization will “release” cytosolic LC3 by flushing away during washing steps. LC3-II trapped in the autophagosome remains intact and fluorescence can be measured.

Autophagy: Four Stages of Autophagy Autophagy can be induced by nutrient depletion or inhibition of mTOR pathway. During autophagy, cytosolic proteins and aging organelles are sequestered by a double membrane vesicle to form autophagosomes. One of the hallmarks of autophagy is translocation of LC3 from the cytoplasm to the autophagosome. Autophagosome then fuses with the lysosome to cause the breakdown of autophagosome vesicle and its contents, including LC3. This process can be visualized using either an GFP-LC3 fusion protein or and anti-LC3 antibody.