Np 40

After the cells were prepared, the medium was absorbed and discarded, washed twice with PBS, and the cells were scraped off with 400 μL precooled PBS. Then, transferred into 1.5 ml tube, centrifuged at 10,000 rpm at 4 °C for 10 s, and the supernatant was discarded. Resuspend the cells with 400 μL PBS containing RNA enzyme inhibitor (N2615, Promega, USA) (RNA enzyme inhibitors: PBS = 1:20 v/v ratio) and 0.1% NP-40 (85124, Thermo Fisher Scientific, USA) (NP-40: PBS = 1:1000 v/v ratio), put it in ice for 5 min, then vortexed for 5 s. After centrifugation at 10,000 rpm at 4 °C for 20 s, the supernatant was extracted from cytoplasm and transferred into a new tube. At this time, the supernatant was cytoplasm extract, and the precipitate was nucleus. The nuclei were resuspended by 200 μL PBS containing 0.5% NP-40 and RNA enzyme inhibitor (RNA enzyme inhibitors: PBS = 1:20 v/v ratio), and were blown for 10 times, placed on ice for 10 min, and centrifuged for 20 s at 13,000 rpm at 4 °C. The supernatant was the nuclear extract, and nuclear extract and cytoplasmic extract were used for subsequent RT-qPCR experiments according to the above steps.

Cells were harvested and the pellets were resuspended in a RIPA buffer (100 mM tris–HCl (pH = 7.4), 0.5% NP-40 (Fisher scientific, Illkirch, France), 0.5% sodium-deoxycholate (Sigma-Aldrich), 0.1% SDS supplemented with proteases inhibitor Mini^® (Roche Diagnostics, Basel, Switzerland)) for 20 min. The solubilized proteins were then recovered in the supernatant after a 20 min-centrifugation at 12,000× g.

Dulbecco’s modified Eagle’s medium (DMEM) 4.5 g/L Glucose and fetal bovine serum (FBS) were from Dominique Dutscher (Brumath, France), Glutamax was from Life Technologies (Milano, Italy), trypsin-EDTA and penicillin/streptomycin were from Biowhittaker (Verviers, Belgium). Triton X-100, N-lauryl sarcosine, MD, Bafilomycin A1, and E64d-protease inhibitors were from SIGMA (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO), NaOH, and Na₂EDTA were from Baker (Deventer, The Netherlands). Tris and NaCl were from Carlo Erba (Milan, Italy). Normal-melting-point agarose, low-melting-point agarose, and ethidium bromide were from Bio-Rad Laboratories (GmbH, Munich, Germany). Nitrocellulose membranes for Western blotting were AmershamProtran™ 0.2 µm NC, Chicago, IL, USA. Antibodies used are rabbit anti-LC3 (#L7543, Sigma Aldrich, St. Louis, MO, USA), anti-ATG5 (A0731, Sigma Aldrich), Anti-ATG7 (clone D12B11, Cell signaling Technology, Danvers, MA, USA), and horseradish peroxidase-conjugated secondary antibodies from Jackson Immunoresearch. NP-40 was from Fisher Scientific (Illkirch, France). Western blots were revealed with Clarity Western ECL (Bio-Rad laboratories, Marnes-la-Coquette, France). Chemiluminescence was detected with a G:Box imaging system (Syngene, Cambridge, UK).