Torin

To measure cell proliferation ability, cells were seeded in 96-well plates at a density of 3 × 10³ per well, and cell growth rate was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit (Roche Diagnostics). The MTT assays in each cell line repeated three times, respectively. Cell proliferation ability was further confirmed by soft agar colony formation assay; 8 × 10³ cells were seeded in 6-well plates, and after 3 weeks of culture cell colonies were counted by crystal violet staining. To detect the effects of inhibitors (selumetinib, rapamycin, torin; Selleck) on cell growth we performed colony-formation assay. After incubation for 3 days with indicated inhibitors, the cells in 12-well plates were fixed with 4% paraformaldehyde (PFA) and stained with 0.1% crystal violet solution to visualize their colony-forming ability.

MEFs from WT and Atg4d-deficient mice were incubated in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) as control and upon two different autophagy-inducing conditions – Torin (Selleckchem, at 250 nM) and EBSS (Sigma-Aldrich)- in a 96 well plate. After 4 h of treatment, CYTO-ID^® detection kit (Enzo Life Science, ENZ-51031) was used to stain the autophagic vesicles according to the product manual. Briefly: treatment media was removed; cells were washed with buffer and the detection solution then added. After 30 min of incubation at 37 °C, cells were washed twice with buffer and immediately analyzed in a BD Pathway 435 System (BD Bioscience).