Figure 2

Quantification of eGFP-LC3B + vesicles using confocal fluorescence microscopy. (A) Schematic overview of the method. eGFP-LC3B expressing cell lines are fixed in 4% PFA and images taken by confocal fluorescence microscopy. Using ImageJ, the area and the count of eGFP-LC3B puncta in the cells can be quantified to measure autophagy. (B) Exemplary confocal images of eGFP-LC3B (green) expressing HeLa, HEK293T, Jurkat, and THP-1 cells. The cells were either mock-treated or treated with Rapamycin (1 µM), Chloroquine (10 µM), or Bafilomycin A1 (2.5 µM) for 4 h before fixation. Nuclei, DAPI (blue). Size marker, 10 µm. (C) Quantification of the eGFP area per cell of HeLa eGFP-LC3B cells, HEK293T eGFP-LC3B, Jurkat eGFP-LC3B, or THP-1 eGFP-LC3B either mock-treated or treated with Rapamycin (1 µM), Chloroquine (10 µM), or Bafilomycin A1 (2.5 µM) for 4 h before fixation, using the method described in A. Results are shown as mean(HeLa GL n = 50–100, HEK293T GL n = 5–6, Jurkat GL n = 6–11, and THP-1 GL n = 10–12) ± SEM, statistical significance was assessed using one-way ANOVA. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.