LC3 puncta quantification

Cells were transiently transfected with GFP-LC3, or co-transfected with pEGFP-N1 and RFP-LC3, or co-transfected with TM9SF4-GFP and RFP-LC3. Twenty-four hours after the transfection, the cells were starved in EBSS for 2 h with or without bafilomycin A1 (10 nM). Fluorescent images were acquired under Laser Scanning Confocal Imaging System (Fluoview 1000; Olympus, Tokyo, Japan). GFP-LC3 puncta or RFP-LC3 puncta per cell were analyzed using ImageJ software.

Similar measurement was used in RFP-GFP-LC3 puncta quantification. Briefly, the cells carrying scrambled-shRNA or TM9SF4-shRNA were further transfected with RFP-GFP-LC3. Twenty-four hours after transfection, the cells were starved in EBSS for 2 h. GFP/RFP-LC3 puncta per cell were quantified by ImageJ software. The number of yellow puncta (both GFP and RFP) represented autophagosome while red puncta (RFP only) represented autolysosomes.