An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication

Mauthe, Mario; Langereis, Martijn; Jung, Jennifer; Zhou, Xingdong; Jones, Alex; Omta, Wienand; Tooze, Sharon A.; Stork, Björn; Paludan, Søren Riis; Ahola, Tero; Egan, Dave; Behrends, Christian; Mokry, Michal; de Haan, Cornelis; van Kuppeveld, Frank; Reggiori, Fulvio

The protocol is identical to the one already described, with few modifications . U2OS cells grown in six-well plates were transfected for 48 h with control siRNA or siRNA targeting ATG13, ULK1, or FIP200. Subsequently, the medium was exchanged with the labeling medium (DMEM and 10% dialyzed FCS containing 0.2 µCi/ ml [ 14 C]-valine + 65 µM valine). After 18 h, the cells were placed into chase medium (DMEM, 10% FCS, and 2 mM valine) and incubated for additional 4 h to allow degradation of short-lived proteins. Cells were then transferred for 2 h in the EBSS medium to induce autophagy or in DMEM containing 10% FCS (no autophagy stimulation). The culture media were collected, and TCA was added to 10%. After centrifugation at 2,000 rpm for 10 min, the radioactivity in the soluble fraction (SF) was measured by scintillation counting. In parallel, cells were lysed in PBS containing 1% Triton-X and TCA was added to the lysate to 10% before freezing the samples overnight at −20°C. The radioactivity of TCA-soluble fraction (cytosol) as well as that of the TCA-insoluble pellet (pellet) dissolved in Solvable solution (PerkinElmer) was then measured after centrifugation at 2,000 rpm for 10 min. Protein degradation was determined as follows: (counts in SF + counts in cytosol)/ (counts in SF + counts in cytosol + counts in pellet).