GFP-LC3 and RFP-GFP-LC3 autophagy analyses

Cytoplasmic puncta formation by otherwise soluble GFP-fused LC3 protein is used as a method to quantify autophagy activation under a fluorescence microscope [26]. Briefly, cells were cultured on top of poly-L-Lys coated coverslides. 48 hours after transfection, cells were fixed with ice-cold 4% PFA in PBS for 30 min. GFP-LC3 dot analysis was performed. Coverslides were analyzed under fluorescence microscope (Olympus BX60, Japan) under 60x magnification. Minimum 150 cells per condition were analyzed. Cells containing more than 15 dots were considered as autophagy positive. Percentage of GFP- LC3 positivity was expressed as a percentage of GFP-LC3 dot positive cells within the total transfected cell population.

RFP-GFP-LC3 dot numbers were determined under a fluorescence microscope (Olympus BX60, Japan) using 60x magnification. Dot numbers were analyzed in 50 cells for each experimental point. Autophagosomes gave both RFP and GFP signals, while autolysosomes were defined as RFP positive dots. Number of autolysosomes was calculated by subtracting GFP positive dot numbers from RFP positive dot numbers.