8 wells micro slides

Mitochondria visualization in living cells was performed using TMRE (tetramethyl rhodamine ethyl ester) (#87917 Sigma) labelling. TMRE is a cationic dye that is rapidly accumulated by mitochondria. 10⁵ cells/well were seeded in 8-wells micro-Slides (#80826, IBIDI, Munich, Germany). At different time points cells were loaded for 20 min at 37 °C with 1 nM TMRE in culture medium and analyzed by epifluorescence microscopy. The acquired images were analyzed with ImageJ software. The network status of the cell was visually characterized as normal if filamentous mitochondria were the only observed mitochondria, intermediate if filamentous mitochondria and puncta mitochondria coexisted and altered if only puncta were present.

Autophagy flux was investigated using the reporter plasmid pBABE-puro-mCherry-EGFP-LC3B (Addgene) as reported previously^{44 (link)}. Briefly, PC3 cells were transfected for 16 hours with 1 μg of plasmid per well in 6-well plates. Then, the cells were trypsinized and seeded in 8-wells micro-Slides (IBIDI). After attachment the cells were treated with propranolol, 2DG or cultured in low glucose condition for the indicated times. The images were acquired by epifluorescence microscopy after 24 and 48 hours of treatment and the ratio between early (red and green) and late autophagosomes (red only) was quantified with the ImageJ software. The analyses were performed on stacked images of several focal planes.