mTOR inhibitors lower an intrinsic barrier to virus infection mediated by IFITM3

Shi, Guoli; Ozog, Stosh; Torbett, Bruce E.; Compton, Alex A.

wash buffer (PBS + 0.1% saponin + 1% BSA). Cells were stained with anti-IFITM2/3 (Proteintech, 66081-1-Ig), and anti-LAMP1 Antibody (Thermo Fisher, PA1-654A) overnight at 4°C. Slides were washed three times and incubated for one hour with Alexa Fluor-conjugated secondary antibodies for one hour at room temperature. Slides were mounted for three days with ProLong Gold Antifade Reagent (Thermo Fisher, P36930). Confocal microscopy analysis of endosomal markers was performed using a Zeiss 710 laser scanning confocal microscope at 64X oil-immersion magnification at a 2-fold digital zoom with a 1024 x 1024 field size. For establishing voltage and aperture parameters, snap images were captured for each channel using control slides stained only with secondary antibody. Images were collected in 16-slice intervals and 0.2 µm apart. At least 50 cells were imaged for each condition. Z-stacks were analyzed with Imaris (Bitplane) using the ImarisCell analysis module to quantify number and intensity of IFITM2/3 and LAMP-1+ vesicles. Statistical analysis of vesicle quantity and staining intensity was conducted using Prism 7 (GraphPad) and evaluated with the Kruskal-Wallis test using Dunn's multiple comparison correction.