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

A minimum of 400 cells per experiment was analyzed using the Cellomics SpotDetector V3 algorithm to determine the mean number of p62 and LC3 puncta per cell as well as the mean number of LC3 puncta area per cell. The Hoechst channel was used to set the autofocus; LC3 puncta were detected using the FITC filter, and p62 puncta were detected using the TRI TC filter. An equal fixed exposure time was automatically set for all the samples, and LC3 and p62 puncta were detected in a cell area that excluded the nucleus. The numbers of nuclei (valid object count), LC3 and p62 puncta per cell (spot per cell), and LC3 and p62 puncta area per cell (spot area per cell) were counted using this approach. To assess cell infection rate, cells were stained with either anti-capsid antibody when infected with EMCV or with anti-VP1 or anti-2bc antibodies, when infected with CV, before to image 5-30 fields of cells per sample using the Hoechst, FITC, or TRI TC filters. The percentage of EMCV-or CV-positive cells was determined by analyzing the collected images using the cell counter application in ImageJ.