Primary human fibroblasts and SHSY-5Y neuroblastoma cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) containing 25 mM glucose, 1 mM pyruvate supplemented with 10% fetal bovine serum (FBS, Gibco), 5% penicillin and streptomycin (P/S, Gibco), 2 mM GlutaMAX (Gibco), 5% CO2, at 37°C. All the cell lines were regularly confirmed free of mycoplasma, using the Venor Gem Classic Mycoplasma PCR Detection Kit (Minerva Biolabs).
For microscopy experiments, ∼4700 fibroblasts/well were seeded on 96-well black microplates (Ibidi). After at least 24 h, cells were treated with the various reagents as follows: 2.5 µM U18666A (U18) 48 h (Abcam), 50 µM chloroquine diphosphate salt (CLQ) 6 h (Sigma) and 100 nM rapamycin/sirolimus (Rapa) for 24 h (Acofarma), as described in the main text and figure legends.
For immunoblotting experiments, cells were grown in 25 cm2 flasks until high confluency was obtained and—where indicated—treated with 50 µM CLQ for 6 h (Sigma) to block autophagic flux.
For microscopy experiments, ∼4700 fibroblasts/well were seeded on 96-well black microplates (Ibidi). After at least 24 h, cells were treated with the various reagents as follows: 2.5 µM U18666A (U18) 48 h (Abcam), 50 µM chloroquine diphosphate salt (CLQ) 6 h (Sigma) and 100 nM rapamycin/sirolimus (Rapa) for 24 h (Acofarma), as described in the main text and figure legends.
For immunoblotting experiments, cells were grown in 25 cm2 flasks until high confluency was obtained and—where indicated—treated with 50 µM CLQ for 6 h (Sigma) to block autophagic flux.