Journal: bioRxiv

Article Title: IFITM1 and IFITM3 cooperate to restrict virus entry in endolysosomes

doi: 10.1101/2025.06.01.657267

Figure Lengend Snippet: Endogenous IFITM1 and IFITM3 co-reside in endolysosomal membranes and the localization of IFITM1 to endolysosomes is IFITM3-dependent. (A) HeLa cells were transfected with control siRNA or siRNA targeting IFITM3 for 48 hours and subsequently treated with 100 units IFNb for 18 hours or left untreated. Cells were stained with Lysotracker, fixed and permeabilized, and immunostained with anti-IFITM1 and anti-IFITM3 followed by confocal immunofluorescence microscopy. Scale bar = 15 microns. (B) Colocalization between IFITM1 and Lysotracker in untreated cells was quantified by Pearson Correlation Coefficient and plotted as mean plus standard error. Symbols represent fields of view containing 5-12 cells. Differences that were statistically significant between the indicated conditions as determined by student’s t test are indicated by (*) ( p < 0.05). (C) Colocalization between IFITM1 and Lysotracker in cells treated with 100 units IFNb was quantified by Pearson Correlation Coefficient and plotted as mean plus standard error. Symbols represent fields of view containing 5-12 cells. Differences that were statistically significant between the indicated conditions as determined by student’s t test are indicated by (*) ( p < 0.05).

Article Snippet: For VSV-HA pseudovirus infection experiments, HeLa cells were transfected with 40 nM Silencer Select siRNA (Thermo Fisher) (Negative Control #1 (4390844), 20 nM IFITM1 (4392421; s16192), 20 nM IFITM3 (4392421; s195035)) or 20 nM IFITM1 (4392421; s16192) plus 20 nM IFITM3 (4392421; s195035) using Lipofectamine RNAiMAX according to manufacturer’s instructions. siRNA and RNAiMAX were diluted in OptiMEM Reduced Serum Medium (Gibco).

Techniques: Transfection, Control, Staining, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: IFITM1 and IFITM3 cooperate to restrict virus entry in endolysosomes

doi: 10.1101/2025.06.01.657267

Figure Lengend Snippet: Endogenous IFITM1 and IFITM3 restrict HA-mediated virus entry. (A) HeLa cells were transfected with control siRNA, siRNA targeting IFITM3, or siRNA targeting IFITM3 plus siRNA targeting IFITM1 for 72 hours and inoculated with replication-incompetent VSV-HA pseudovirus. Infection was measured by GFP expression at 18 hours post-inoculation and plotted as mean plus standard error. Symbols represent three independent experiments (biological replicates). Differences that were statistically significant between the indicated conditions as determined by one-way ANOVA are indicated by (*) ( p < 0.05). (B) HeLa cells were transfected with control siRNA, siRNA targeting IFITM1, siRNA targeting IFITM3, or siRNA targeting IFITM1 plus siRNA targeting IFITM3 for 72 hours. Cells were lysed and whole cell lysates were subjected to SDS-PAGE and immunoblotting with anti-IFITM1, anti-IFITM3, and anti-Actin (used as loading control). Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. GFP; green fluorescent protein. VSV; vesicular stomatitis virus. HA; hemagglutinin.

Article Snippet: For VSV-HA pseudovirus infection experiments, HeLa cells were transfected with 40 nM Silencer Select siRNA (Thermo Fisher) (Negative Control #1 (4390844), 20 nM IFITM1 (4392421; s16192), 20 nM IFITM3 (4392421; s195035)) or 20 nM IFITM1 (4392421; s16192) plus 20 nM IFITM3 (4392421; s195035) using Lipofectamine RNAiMAX according to manufacturer’s instructions. siRNA and RNAiMAX were diluted in OptiMEM Reduced Serum Medium (Gibco).

Techniques: Virus, Transfection, Control, Infection, Expressing, SDS Page, Western Blot