Journal: Journal of Virology

Article Title: SARS-CoV-2 Variants of Concern Hijack IFITM2 for Efficient Replication in Human Lung Cells

doi: 10.1128/jvi.00594-22

Figure Lengend Snippet: Impact of IFITMs on replication of the Omicron VOC. (A) Schematic presentation of amino acid variations in Spike proteins of the SARS-CoV-2 Delta and Omicron VOCs investigated. Abbreviations are defined in the legend to . (B) Viral N RNA levels in the supernatant of Calu-3 cells infected with the indicated SARS-CoV-2 variants. Cells were transfected with control or IFITM-targeting siRNAs as indicated. Numbers above the bars indicate n -fold reduction compared to the viral RNA levels detected in the supernatant of Calu-3 cells treated with control siRNA. Bars in panels B and C show the mean of 3 independent experiments (±SEM), each measured in technical duplicates. (C) Quantification of intracellular viral N RNA levels in Calu-3 cells 24 h postinfection with SARS-CoV-2 (MOI of 0.05). Values were normalized to GAPDH and calculated relative to the control (set to 100%). Cells were transiently transfected with siRNA—either control (CTRL) or targeting IFITM1, -2, or -3. (D) Exemplary Western blot of IFITM and viral N protein expression in Calu-3 cells treated with control or IFITM1-, -2-, and/or -3-targeting siRNA and infected with the indicated SARS-CoV-2 variants. GAPDH was detected as a loading control. P values: *, <0.05; **, <0.01; ****, <0.0001.

Article Snippet: Proteins were stained at 1:1,000 using primary antibodies against IFITM1 (Cell Signaling, catalog no. 13126S), IFITM2 (Cell Signaling, catalog no. 13530 S), IFITM3 (Cell Signaling, catalog no. 59212S), ACE2 (rabbit polyclonal) (Abcam, catalog no. ab166755), rat anti-GAPDH (Biolegend, catalog no. 607902), and SARS CoV-2 N (Sino Biologicals, catalog no. 40588-V08B) and infrared dye-labeled secondary antibodies (Li-Cor IRDye).

Techniques: Infection, Transfection, Control, Western Blot, Expressing

Journal: Journal of Virology

Article Title: SARS-CoV-2 Variants of Concern Hijack IFITM2 for Efficient Replication in Human Lung Cells

doi: 10.1128/jvi.00594-22

Figure Lengend Snippet: Expression of ACE2 and IFITM proteins in Calu-3 and iATII cells. (A) Immunoblot of ACE2, IFITM1, IFITM2, and IFITM3 in Calu-3 and iATII cells left uninfected (lanes C) or infected with the indicated SARS-CoV-2 variants. Whole-cell lysates were stained with the indicated antibodies. An unspecific signal was observed in the Calu-3 control lane stained with the CoV-2 N antibody. (B) Flow cytometric analysis of surface ACE2 expression in Calu-3 and iATII cells.

Article Snippet: Proteins were stained at 1:1,000 using primary antibodies against IFITM1 (Cell Signaling, catalog no. 13126S), IFITM2 (Cell Signaling, catalog no. 13530 S), IFITM3 (Cell Signaling, catalog no. 59212S), ACE2 (rabbit polyclonal) (Abcam, catalog no. ab166755), rat anti-GAPDH (Biolegend, catalog no. 607902), and SARS CoV-2 N (Sino Biologicals, catalog no. 40588-V08B) and infrared dye-labeled secondary antibodies (Li-Cor IRDye).

Techniques: Expressing, Western Blot, Infection, Staining, Control

Journal: Clinical Cancer Research

Article Title: IFN-Induced Transmembrane Protein 1 Promotes Invasion at Early Stage of Head and Neck Cancer Progression

doi: 10.1158/1078-0432.ccr-07-4761

Figure Lengend Snippet: Fig. 2. IFITM1promotes the invasion of HNSCC cells. A, generation of IFITM1-overexpressing cells. Ca9-22 cells were engineered to overexpressing IFITM1by transfection with pBICEP ^ CMV-2^ IFITM1.We obtained three stable clones of IFITM1-overexpressing cells. Ectopic expression of IFITM1was examined by immunoblotting with anti-FLAG antibody.The whole lysates from all samples were blotted with Cul1for a loading control. B, cell proliferation of IFITM1-overexpressing HNSCC cells. Cells were plated on 24-well plates, and trypsinized cells were counted by Cell Counter at 4 d. C, invasion of IFITM1-overexpressing HNSCC cells.The invasiveness of the cells was determined by in vitro invasion assay.1.5 105 cells were placed in the upper compartment of the cell culture insert for 24 h.To examine the invasiveness, penetrated cells onto the lower side of the filter were fixed with formalin and stained with hematoxylin.We assayed thrice. *, P < 0.05 when compared with parent cells. D, migration of IFITM1-overexpressing HNSCC cells.We used one of stable clones for wound healing assay. Migration of the cells was determined by wound healing assay. At 24 h after scratching the cells, phase-contrast images (10 field) of the wound healing process were photographed digitally with an inverted microscope.The distance of the wound areas was measured on the images, set at 100% for 0 h, and the mean percentage of the total distances of the wound areas was calculated. *, P < 0.05 when compared with control cells.

Article Snippet: An anti-IFITM1 polyclonal antibody (Boster Biological Technology Ltd.), anti-CD81 monoclonal antibody (BD Biosciences), anti-FLAG monoclonal antibody (Sigma), and antiCul1 polyclonal antibody (Zymed) were used.

Techniques: Transfection, Clone Assay, Expressing, Western Blot, Control, In Vitro, Invasion Assay, Cell Culture, Staining, Migration, Wound Healing Assay, Inverted Microscopy

Journal: Clinical Cancer Research

Article Title: IFN-Induced Transmembrane Protein 1 Promotes Invasion at Early Stage of Head and Neck Cancer Progression

doi: 10.1158/1078-0432.ccr-07-4761

Figure Lengend Snippet: Fig. 4. Correlation between IFITM1and CD81in HNSCC cells. A, expression of CD81mRNA in six HNSCC cell lines, normal oral epithelial cells, and gingival fibroblasts. Expression of CD81mRNA was done by RT-PCR.The picture of IFITM1and GAPDH mRNA expression is same as Fig.1B. B, Expression of CD81protein in 6 HNSCC cell lines by immunoprecipitation and immunoblotting of CD81. CD81was precipitated from lysates with the use of monoclonal antibodies specific CD81and immunoblots was probed with CD81and IFITM1antibodies. IgGLch, IgG light chain; IP, immunoprecipitation; and IB, immunoblotting. C, CD81expression was examined in IFITM1-overexpressing and IFITM1knockdown cells by RT-PCR.The picture of IFITM1and GAPDH expression in IFITM1-overexpressing and IFITM1knockdown cells is same as Figs. 2A and 3A, respectively.Top, CD81expression in stable clones of IFITM1-overexpressing cells; bottom, CD81expression in IFITM1shRNA-treated cells.

Article Snippet: An anti-IFITM1 polyclonal antibody (Boster Biological Technology Ltd.), anti-CD81 monoclonal antibody (BD Biosciences), anti-FLAG monoclonal antibody (Sigma), and antiCul1 polyclonal antibody (Zymed) were used.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Western Blot, Bioprocessing, Clone Assay

Journal: Clinical Cancer Research

Article Title: IFN-Induced Transmembrane Protein 1 Promotes Invasion at Early Stage of Head and Neck Cancer Progression

doi: 10.1158/1078-0432.ccr-07-4761

Figure Lengend Snippet: Fig. 5. CD81is involved in the invasion of HNSCC cells. A, three different CD81shRNAs were stably transfected in HSC2 cells. After transfection, we treated blasticidin for selection. CD81and IFITM1mRNA expression is examined by RT-PCR. GAPDH expression was used as a loading control. B, cell proliferation of CD81shRNA-treated cells (top). Cells were plated on 24-well plates, and trypsinized cells were counted by Cell Counter at 0 and 4 d.We defined 1as a number of cells at 0 d and calculated the fold change of number of cells at 4 d. Invasion of CD81shRNA-treated cells (bottom).The invasiveness of the cells was determined by in vitro invasion assay.1.5 105 cells were placed in the upper compartment of the cell culture insert for 24 h.To examine the invasiveness, penetrated cells onto the lower side of the filter were fixed with formalin and stained with hematoxylin.We assayed thrice. *P < 0.05 when compared with parent cells. C, we examined the cotransfection of CD81shRNA into IFITM1transfected HNSCC cells.Three different CD81shRNAs (1, 2, and 3) were transiently transfected into IFITM1-overexpressing Ca9-22 cells. IFITM1and CD81expression was examined by RT-PCR (top). GAPDH was used as a control.The invasiveness of the cells was determined by in vitro invasion assay (bottom).1.5 105 cells were placed in the upper compartment of the cell culture insert for 36 h.To examine the invasiveness, penetrated cells onto the lower side of the filter were fixed with formalin and stained with hematoxylin.We assayed thrice. *P < 0.05 when compared with IFITM1overexpressing cells without shCD81translation. D, IFITM1is not induced byWnt/h-catenin signaling pathway.We treated h-Trcp1/2 siRNA in HNSCC cells. A 21-nt duplex corresponding to a nonrelevant F-box protein gene was used as a control. h-Trcp1/2 siRNA induced down-regulation of h-Trcp1and h-Trcp2 mRNA and accumulation of h-catenin protein. In this h-Trcp1/2 siRNA-treated cells, IFITM1mRNAwas examined by RT-PCR. GAPDH was used as a control for RT-PCR. Cul1was used as a loading control forWestern blot analysis. E, gene expression profiles of control and IFITM1-overexpressing HNSCC cells. ZNF236, MMP13, and MMP12 were up-regulated in IFITM1-overexpressing cells (Table1).We confirmed the expression of ZNF236, MMP13, and MMP12 mRNA in IFITM1HNSCC-overexpressing cells by RT-PCR. GAPDH expression was used as a loading control.

Article Snippet: An anti-IFITM1 polyclonal antibody (Boster Biological Technology Ltd.), anti-CD81 monoclonal antibody (BD Biosciences), anti-FLAG monoclonal antibody (Sigma), and antiCul1 polyclonal antibody (Zymed) were used.

Techniques: Stable Transfection, Transfection, Selection, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, In Vitro, Invasion Assay, Cell Culture, Staining, Cotransfection, Gene Expression