Nunc flasks

The specificity of the anti-LC3b (rabbit polyclonal; Abcam plc; no. ab48394, dilution 1:200) antibody was tested using LnCaP prostate cancer cells (American Type Culture Collection, Manassas, VA). LnCaP cells were cultured in the presence of pharmacological modulator of autophagy, 5 mM 3-MA (Sigma-Aldrich, Buchs, Switzerland), an inhibitor commonly used to block autophagy [35 (link)] and 2 μM rapamycin (LuBioScience, Luzern, Switzerland) an inducer of autophagy for 7 days on the slide flasks (Nunc flasks, cat: 170920). Cells were immunostained with anti-LC3b (1:200) for 24h at 4°C. The slides were incubated with secondary antibody Cy3-conjugated sheep anti-rabbit antibody (Sigma, 1:200) at room temperature for 1 h. The slides were counter-stained with DAPI (4′,6-diamidino-2-phenylindole, Sigma, 1:200) and analysed with a Leica fluorescence microscope (40x). The specificity of the anti-LC3b antibody could be confirmed by detecting no staining in the negative control, a minimal staining in cells inhibited by 3MA, a modest staining in untreated cells (basal autophagy level) and the typical LC3b punctuation in cells induced by rapamycin (Supplementary Figure 1).

PANC-1 cells were cultured in DMEM high glucose (25 mmol/L) supplemented with 10% FBS (PANC-1 medium) according to the manufacturer’s instructions. Cells were kept in 75 cm² Nunc™ flasks before being cultured for experiments and then kept in a humidified in a 5% CO₂ atmosphere at 37 °C, and the medium was changed every 2–3 days.