IFITM3 Clusters on Virus Containing Endosomes and Lysosomes Early in the Influenza A Infection of Human Airway Epithelial Cells

Kummer, Susann; Avinoam, Ori; Kräusslich, Hans-Georg

A549 and MDCK cells were maintained in Dulbecco's modified Eagle Medium (DMEM) (Invitrogen, Karlsruhe, Germany), supplemented with 10% foetal calf serum (FCS) (Invitrogen) and penicillin (200 U/mL)/streptomycin sulphate (100 µg/mL) (Capricorn scientific GmbH, Ebsdorfergrund, Germany). Human small airway epithelial cells (HSAEpCs) were obtained from PromoCell (C-12642) and grown in a primary cell basal medium (PromoCell) without antibiotics. Four days after thawing, the HSAEpCs were seeded for immunofluorescence. The IFITM3 knock-down in A549 cells was induced by CRISPR-Cas9 gene editing using a single guide RNA targeting the polyadenylation side of IFITM [59, 60] . A549 IFITM3 knock-down cells were selected by puromycin resistance (10 µg/mL). The IFITM3 expression was checked by real-time quantitative PCR, as described by Zhao et al., 2013 [61] using the 7500 Fast Real Time PCR System (Applied Biosystems, Lifetechnologies, sigma-aldrich, Steinheim, Germany) and by immunofluorescence, as noted below ( Figure S5 ).