Spe 2

3 × 10⁵ dTHP-1 cells were seeded on 8 mm coverslips in 24 wells plates and differentiated for 48 h with 100 nM PMA in 0.5 ml medium. Wells were washed 3 times, and cells were primed for 3 h with TAMRA-labelled DCATH-2 (TD) or medium. For inhibition conditions, cells were pre-incubated before priming for 1 h with 3 µM KN-62, 50 µM suramin, 10 µM chlorpromazine, or 10 µg/ml nystatin. After priming, coverslips were washed 3 times with warm medium followed by fixation for 30 min in 4% paraformaldehyde solution (0.1 M phosphate buffer, pH 7.4). After staining of nuclei with Hoechst (Molecular probes, Eugene, OR, USA) for 10 min, coverslips were mounted on glass slides using ProLong glass antifade mountant (Thermofisher scientific, Waltham, MA, USA). Confocal images were acquired on a Leica SPE-II using the 100x HCX PLAN APO (NA = 1.4-0.7) objective. Imaging was performed using a quadruple band beam splitter for the 561 nm laser. Visualization of Hoechst stained dTHP-1 cells was done with a 405 nm (100 mW) Coherent Violet Cube laser. Image analysis was done using composite images of both channels. In brief, regions of interest were set manually using DIC for segmentation. To isolate puncta from background noise, a threshold was set for the TAMRA channel and intensity and area of puncta were measured partially automated.

To image the localization of LAMP1 in HAP1, H1-ΔV33, and H1-ΔV33A-fV33, cells the cells were seeded onto coverslips one day prior to staining. Cells were fixed in 4% formaldehyde in PBS for 15 min at RT, washed with PBS, and subsequently permeabilized in PBS containing 0.1% Triton-X-100 for 10 min. Cells were incubated with antibody against LAMP1 (rabbit anti-LAMP1 pAb, 1∶100 dilution; Abcam) in 3% BSA in PBS followed by incubation with secondary antibodies coupled to AF488, AF-568 phalloidin, and DAPI (all Life Technologies). The samples were analyzed using a confocal laser-scanning microscope (Leica SPE-II).

Cells were incubated with virus for 1 h at 37°C, supplied with fresh medium, and incubated at 37°C for 6 h (luciferase assays) or 7 h (immunofluorescence). In neutralization assays, viruses or cells were pretreated with ligands for 1 h at 37°C, unless otherwise indicated. Crystal violet staining was performed 2 days postinfection. For immunofluorescence staining, cells were fixed by submersion in a 4% paraformaldehyde solution for 15 min. Fixed cells were stained with 1:1,000 diluted rabbit antiserum against mengovirus capsids (obtained from Ann Palmenberg, University of Wisconsin) or a 1:100 diluted mouse monoclonal antibody against CV-B3 protein 3A. Cells were examined by confocal microscopy (Leica SPE-II), and the number of infected cells was quantified with ImageJ.

IFA was performed as described previously [18 (link)]. Briefly, cells were grown on 12-mm glass coverslips and the next day infected with the viruses indicated above (MOI=10 or 50). At the indicated time points, cells were fixed in 4% paraformaldehyde for 30 min and then permeabilized using 0.1% Triton X-100. Samples were incubated with primary antibody for 1 h and incubated with secondary antibodies and DAPI for 30 min. Coverslips were mounted on microscopy slides with FluorSafe^TM reagent (Merck Millipore, Massachusetts, USA). Finally, cells were examined by confocal microscopy (Leica SPE-II, Wetzlar, Germany) and Leica Application Suite Advanced Fluorescence (LAS-AF, Wetzlar, Germany) software.