Quantification of Autophagy Levels

Autophagy quantification was performed as described in Jacomin et al. [26 (link)]. Briefly, HeLa GFP-LC3 cells were seeded on 96-well plate (#655090, Greiner) at 7500 cells per well; 24 h later, cells were treated with DMSO at 0.25% and rapamycin at 250 mM, as bioinactive and bioactive controls, respectively, or with the extracts at the indicated dilution, for 2 h. After fixation (4% paraformaldehyde), the DNA was stained with 1 µg/mL Hoechst. Image acquisitions were performed on an ArrayScan^VTI using a Zeiss 20× Plan-Neoflur air objective (10 fields per well). Images were automatically analyzed with SpotDetector Bio-Application of Thermo Scientific (Illkirch, France) HCS Studio v6.5.0, allowing us to count and extract the GFP-LC3 dots per cell parameter. Results are presented as a proportion of the cells containing less than three autophagosomes (ATG), between three and eight, and more than eight ATG.