Fig 1: Integration of TAILS results with data from RNAi screens for IAV host factors. (A, B) List of potential direct targets of CTSW for which an antiviral (A) or a proviral (B) role in IAV infection was predicted by Metascape (15). (C) Representative images of HEK293T cells incubated with LysoTracker to label acidic organelles (red) in live cells treated with (w/) or without (w/o) 10 nM bafilomycin A1 (BafA1) for 16 h. DAPI was used to counterstain the nuclei (blue). (D) HEK293T cells cotransfected with HA-EPN2- and CTSW- or GFP-encoding plasmids were treated with DMSO or 40 nM bafilomycin A1 (BafA1) 4 h posttransfection for 20 h. Cell lysates were analyzed by Western blotting using polyclonal rabbit anti-GAPDH, monoclonal mouse anti-EPN2, and monoclonal mouse anti-CTSW antibodies. (E) Sequence alignment of epsin 2 (EPN2) and epsin 1 (EPN1) is shown at the top. The unique EPN2 peptide identified by TAILS (terminal amine isotopic labeling of substrates) and the corresponding peptide for EPN1 are boxed. Western blots analyzing HEK293T cells cotransfected with GFP-EPN1- or GFP-EPN2-expressing plasmids and constructs encoding GFP, CTSW WT, or CTSW mut are shown below. In addition to a monoclonal mouse anti-EPN1 antibody, the same antibodies as described in the legend to panel D were used. (F) A549 cells were transfected with a nontargeting siRNA (siSCR) or siRNAs targeting CTSW (siCTSW) or epsin 1 (siEPN1#2, #4, and #6). At 48 h posttransfection, cells were infected with A/WSN/1933 (MOI = 10 PFU per cell) for 1 h at 4°C, followed by 3 h at 37°C. Fixed and permeabilized cells were then stained for the viral nucleoprotein (NP) and nuclei and analyzed by fluorescence microscopy. Mean fluorescence intensity (MFI) in the nuclei (nuclear NP signal) was quantified using ImageJ (n > 400) and normalized to that of cells treated with siSCR. Mean values from three independent experiments are plotted, error bars represent standard errors of the means (SEMs), and individual data points are shown. (G) Knockdown efficiencies of three siRNAs targeting EPN1 were assessed by reverse transcription-qPCR analysis. GAPDH transcript levels were used for normalization. Shown are mean values from three independent experiments, each performed at least in duplicates. (H) A549-GFP (black symbols) and A549-GFP-EPN2 (pink symbols) cells were infected with WSN-Ren (MOI = 2), and Renilla luciferase expression was monitored with the live-cell substrate EnduRen at the indicated time points. The viral growth curves were measured in three independent experiments, and the area under the curve (AUC) was determined. (F to H) Statistical testing was done using the unpaired t test: ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.