Author: 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
Title: An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication Document date: 2016_8_29
ID: iuqa0yrw_69
Snippet: U2OS, HeLa or HEK293T-REx cells were infected at an MOI of 0.01 with CVB3 or an MOI of 0.1 with EMCV, EV71, EMCV-Zn, or CVA21 in DMEM for 2 h before replacing it with fresh DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS and continuing the incubation for an additional 24-48 h. Supernatants were collected to determine the virus titer while cells were fixed with 3.7% PFA and cell nuclei were stained with Hoechst3334.....
Document: U2OS, HeLa or HEK293T-REx cells were infected at an MOI of 0.01 with CVB3 or an MOI of 0.1 with EMCV, EV71, EMCV-Zn, or CVA21 in DMEM for 2 h before replacing it with fresh DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS and continuing the incubation for an additional 24-48 h. Supernatants were collected to determine the virus titer while cells were fixed with 3.7% PFA and cell nuclei were stained with Hoechst33342 and processed for automated image acquisition and analysis in the following way. The number of cells from four fields per well was determined by counting the number of nuclei using the Cellomics Arrayscan VTI HCS Reader with a 5× objective. The percentage of surviving cells in each sample was calculated by dividing the number of infected cells with that of uninfected cells.
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