Journal: Nature Chemical Biology

Article Title: IFITM3 directly engages and shuttles incoming virus particles to lysosomes

doi: 10.1038/s41589-018-0213-2

Figure Lengend Snippet: a , Alignment and topology of human IFITM1, IFITM2 and IFITM3. Key domains: amphipathic (AP) helix, hydrophobic (HP), and transmembrane (TM); mutations: N21Δ SNP rs12252-C allele and post-translational modifications are highlighted. b , c , Expression levels of human IFITM1, IFITM2 and IFITM3 in IFN-treated HeLa and A549 cell lines. Full gels for b are shown in Supplementary Fig. . d , IAV infection of naïve and IFN-α stimulated HeLa WT and IFITM2/3-KO cell lines. Cells were stimulated with IFN-α (100 μg/mL) for 16 h and then infected with IAV (multiplicity of infection (MOI) = 2.5). At 12 h post-infection, cells were harvested, stained for IAV and analyzed by flow cytometry. The percentage of IAV-positive cells was normalized to WT cells and plotted as relative infection. Data represent the mean and s.d. of three independent experiments. e , Stable reconstitution with IFITM2 and IFITM3 in IFITM2/3-KO HeLa cells (C5-2). Low, medium and high IFITM2/3 protein expression levels are shown. f , IAV infection of IFITM2- and IFITM3-reconstituted HeLa and A549 cell clones stimulated with IFN-α (100 μg/mL). Experiments were performed and data were analyzed as described in d .

Article Snippet: Anti-IFITM3 (polyclonal) 10088-604 was from VWR International.

Techniques: Expressing, Infection, Staining, Flow Cytometry, Clone Assay

Journal: Nature Chemical Biology

Article Title: IFITM3 directly engages and shuttles incoming virus particles to lysosomes

doi: 10.1038/s41589-018-0213-2

Figure Lengend Snippet: a , Localization of IFITM3 and CD63 in IFN-α stimulated A549 cells. Pearson correlation coefficient (PCC) analyses are included below. Top row, endogenous IFITM3 and CD63 in IFN-α stimulated WT A549 cells (PCC = 0.58); middle row, endogenous IFITM3 and CD63 in IFN-α stimulated IFITM2/3-KO cells (PCC = n/a); bottom row, IFITM2/3-KO cells + stable re-expression of IFITM3 (PCC = 0.46). b , EGFR levels in IFN-α stimulated A549 WT, IFITM2/3-KO and IFITM2/3-KO cells + stable re-expression of IFITM3. Cells were treated with IFN-α (100 μg/ml) for 16 h, serum-starved for 2 h, treated with 25 μg/ml cycloheximide for 1 h and then treated with 100 ng/ml EGF for 30 min at 4 °C in the continued presence of cycloheximide. Cells were subsequently shifted to 37 °C and analyzed at various time points. Full gels are shown in Supplementary Fig. . c , Quantitation of EGFR levels in b normalized to tubulin levels. Data represent the mean and s.d. of three independent experiments.

Article Snippet: Anti-IFITM3 (polyclonal) 10088-604 was from VWR International.

Techniques: Expressing, Quantitation Assay

Journal: Nature Chemical Biology

Article Title: IFITM3 directly engages and shuttles incoming virus particles to lysosomes

doi: 10.1038/s41589-018-0213-2

Figure Lengend Snippet: a , IAV lipid mixing in an IFITM3 + compartment over time. Bar, 5 μm. b, The fluorescence intensity trace for the indicated particle in a. c , Example of IAV lipid mixing before colocalization with IFITM3 in HeLa cells. Scale bar, 5 μm. For a and c , the arrows indicate the virion of interest in each panel. The numbers indicate the time post-infection (minutes). d , The fluorescence intensity trace for the indicated particle in c. e , Percentage of particles of IAV, and VSV pseudotyped with LASV or EBOV glycoproteins undergoing lipid mixing in LAMP1 + or IFITM3 + compartments in HeLa cells. Data represent the mean and s.d. of three independent experiments ( n = 800–1,000 cell-associated particles total for each sample). P values were determined by one-way ANOVA with a post-hoc Tukey test. * P < 0.05, *** P < 0.001, ****P < 0.0001. f , Time of lipid mixing relative to IAV, LASV- or EBOV-pseudotyped particle colocalization with IFITM3 in HeLa cells ( n > 50 dequenching particles tracked in three independent experiments for each sample).

Article Snippet: Anti-IFITM3 (polyclonal) 10088-604 was from VWR International.

Techniques: Fluorescence, Infection

Journal: Nature Chemical Biology

Article Title: IFITM3 directly engages and shuttles incoming virus particles to lysosomes

doi: 10.1038/s41589-018-0213-2

Figure Lengend Snippet: a , Western blot analysis of IFITM2 and IFITM2/3 in IFN-α stimulated HeLa WT and IFITM2/3-KO cells. High and low exposures of blots are included. Full gels are shown in Supplementary Fig. . b , IAV infection of IFN-α stimulated HeLa WT and IFITM2/3-KO cells. Data represent the mean and s.d. of three independent experiments. c , ZIKV infection of IFN-α stimulated HeLa WT and IFITM2/3-KO cells. Data represent the mean and s.d. of three independent experiments. d , IAV-DID dequenching and trafficking with IFITM3-N21Δ. The arrows indicate the virion of interest. The numbers indicate the time post-infection (in minutes). Scale bar, 5 μm. e , Fluorescence intensity trace for the indicated particle in d . f , Relative percentage of DiD-IAV particles colocalized with IFITM3 and IFITM3-N21Δ at the time of dequenching. Data represent the mean and s.d. of three independent experiments ( n > 100 dequenching particles total for each sample. P < 0.001 by an unpaired, two-sided t -test).

Article Snippet: Anti-IFITM3 (polyclonal) 10088-604 was from VWR International.

Techniques: Western Blot, Infection, Fluorescence

Journal: Nature Chemical Biology

Article Title: IFITM3 directly engages and shuttles incoming virus particles to lysosomes

doi: 10.1038/s41589-018-0213-2

Figure Lengend Snippet: a , Intensity trace for DiD-IAV dequenching in the presence of IFITM3-F8-BODIPY-C71A. b , Intensity trace for DiD-IAV dequenching in the presence of IFITM3-F8-BODIPY-C72A. c , Intensity trace for DiD-IAV dequenching in the presence of IFITM3-F8-BODIPY-C105A. d , Total percentage of DiD-IAV particles colocalized with IFITM3 Cys mutants at the time of lipid mixing. Data represent the mean and s.d. of three independent experiments ( n > 100 dequenching particles total for each sample). P values determined by one-way ANOVA with a post-hoc Tukey test. * P < 0.05, *** P < 0.001.

Article Snippet: Anti-IFITM3 (polyclonal) 10088-604 was from VWR International.

Techniques:

Journal: Frontiers in Veterinary Science

Article Title: Interferon-Inducible Transmembrane Protein 3-Containing Exosome as a New Carrier for the Cell-to-Cell Transmission of Anti- Brucella Activity

doi: 10.3389/fvets.2021.642968

Figure Lengend Snippet: Lentivirus packaging system.

Article Snippet: The membranes were blocked with 5% non-fat milk (Thermo Scientific, MA, USA) and probed with rabbit anti-phospho-IFITM3 (dilution 1:2,000, EterLife, China) for 1 h at 37°C.

Techniques:

Journal: Frontiers in Veterinary Science

Article Title: Interferon-Inducible Transmembrane Protein 3-Containing Exosome as a New Carrier for the Cell-to-Cell Transmission of Anti- Brucella Activity

doi: 10.3389/fvets.2021.642968

Figure Lengend Snippet: RNA transcription system of reverse transcription-PCR (RT-PCR).

Article Snippet: The membranes were blocked with 5% non-fat milk (Thermo Scientific, MA, USA) and probed with rabbit anti-phospho-IFITM3 (dilution 1:2,000, EterLife, China) for 1 h at 37°C.

Techniques:

Journal: Frontiers in Veterinary Science

Article Title: Interferon-Inducible Transmembrane Protein 3-Containing Exosome as a New Carrier for the Cell-to-Cell Transmission of Anti- Brucella Activity

doi: 10.3389/fvets.2021.642968

Figure Lengend Snippet: List of major protein expression differences in exosomes from host cells.

Article Snippet: The membranes were blocked with 5% non-fat milk (Thermo Scientific, MA, USA) and probed with rabbit anti-phospho-IFITM3 (dilution 1:2,000, EterLife, China) for 1 h at 37°C.

Techniques: Expressing, T-Test, Significance Assay

Journal: Frontiers in Veterinary Science

Article Title: Interferon-Inducible Transmembrane Protein 3-Containing Exosome as a New Carrier for the Cell-to-Cell Transmission of Anti- Brucella Activity

doi: 10.3389/fvets.2021.642968

Figure Lengend Snippet: IFITM3 protein is required for the establishment of host immune response against Brucella . (A) LV1-IFITM3 packaging and infection process. (a): 293T cells; (b): Lentivirus package; (c): RAW264.7 cells; (d): Lentivirus transfection. (B) RT-PCR analysis of relative mRNA expression of IFITM3 in macrophages infected with the siRNA-IFITM3 lentivirus vectors. The 2 −ΔΔCt method was used to calculate relative gene expression. (C) Brucella intracellular growth was determined after 4, 12, 24, and 48 h post-infection by CFU test. (D) Macrophages pretreated with IFITM3 siRNA, siNC, or GW4869 were infected with Brucella M5 and were lysed after 24 h post-infection. The IFITM3 in cell lysate was analyzed by Western blotting. All treatments were repeated three times with n = 3/time point. Statistical significance is indicated by * p < 0.05.

Article Snippet: The membranes were blocked with 5% non-fat milk (Thermo Scientific, MA, USA) and probed with rabbit anti-phospho-IFITM3 (dilution 1:2,000, EterLife, China) for 1 h at 37°C.

Techniques: Infection, Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

Journal: Frontiers in Veterinary Science

Article Title: Interferon-Inducible Transmembrane Protein 3-Containing Exosome as a New Carrier for the Cell-to-Cell Transmission of Anti- Brucella Activity

doi: 10.3389/fvets.2021.642968

Figure Lengend Snippet: Identification of IFITM3 in exosomes secreted from Brucella M5-infected cells. (A) Macrophages were infected with Brucella M5 for 6, 12, and 24 h, and exosomes in the cell culture media were extracted. Western blot analysis of IFITM3 protein in exosome. (B) Macrophages pretreated with IFITM3 siRNA, siNC, or GW4869 were infected with Brucella M5, and exosomes derived from each group were extracted. Western blot analysis of IFITM3 protein in exosome. (C) Macrophages were infected with Brucella M5 at different MOI, and exosomes in this cell culture medium were extracted. Western blot analysis of IFITM3 protein in exosomes.

Article Snippet: The membranes were blocked with 5% non-fat milk (Thermo Scientific, MA, USA) and probed with rabbit anti-phospho-IFITM3 (dilution 1:2,000, EterLife, China) for 1 h at 37°C.

Techniques: Infection, Cell Culture, Western Blot, Derivative Assay

Journal: Frontiers in Veterinary Science

Article Title: Interferon-Inducible Transmembrane Protein 3-Containing Exosome as a New Carrier for the Cell-to-Cell Transmission of Anti- Brucella Activity

doi: 10.3389/fvets.2021.642968

Figure Lengend Snippet: Exosomes released from Brucella M5-infected cells transfer IFITM3 to recipient cells. (A) Macrophages were incubated with purified 30 μg/ml of Exo-M5-IFITM3, Exo-M5-siIFITM3, or Exo for 0, 6, 12, and 24 h. Western blot analysis of IFITM3 protein in these cell groups. (B) siIFITM3-Mø cells were incubated with different doses of purified exosomes (Exo-M5-IFITM3, Exo-M5-siIFITM3, and Exo-M5-siNC), Western blot analysis of IFITM3 protein in these cell groups. (C) The transcription level of IFITM3 in normal macrophages from each group as analyzed by RT-PCR; the expression levels of this protein were normalized to that of GAPDH.

Article Snippet: The membranes were blocked with 5% non-fat milk (Thermo Scientific, MA, USA) and probed with rabbit anti-phospho-IFITM3 (dilution 1:2,000, EterLife, China) for 1 h at 37°C.

Techniques: Infection, Incubation, Purification, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Frontiers in Veterinary Science

Article Title: Interferon-Inducible Transmembrane Protein 3-Containing Exosome as a New Carrier for the Cell-to-Cell Transmission of Anti- Brucella Activity

doi: 10.3389/fvets.2021.642968

Figure Lengend Snippet: The anti- Brucella activity of IFITM3-laden exosomes. siFITM3-Mø cells incubated with PBS, Exo-M5-siIFITM3, Exo-M5-IFITM3, or Exo-M5-siNC were infected with Brucella M5. The CFU level was measured at different time points post-infection. (A) The intracellular bacterial survival was tested by measuring CFU, with PBS as a control group. The macrophage culture supernatants were harvested and IFN-α (B) and IFN-β (C) production (pg/ml) were assessed using the ELISA assay. All treatments were repeated three times with n = 3/time point. Statistical significance is indicated by * p < 0.05.

Article Snippet: The membranes were blocked with 5% non-fat milk (Thermo Scientific, MA, USA) and probed with rabbit anti-phospho-IFITM3 (dilution 1:2,000, EterLife, China) for 1 h at 37°C.

Techniques: Activity Assay, Incubation, Infection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Veterinary Science

Article Title: Interferon-Inducible Transmembrane Protein 3-Containing Exosome as a New Carrier for the Cell-to-Cell Transmission of Anti- Brucella Activity

doi: 10.3389/fvets.2021.642968

Figure Lengend Snippet: Mice immunized with exosomes carrying IFITM3 were protected against Brucella infection. Mice immunized with different groups of exosomes were infected with Brucella M5 for 1, 3, 5, and 7 weeks infection. Then, each group of mice was sacrificed, and mice spleen index (A) and spleen CFU (B) were measured. All treatments were repeated three times with n = 3/time point. Statistical significance is indicated by * p < 0.05, ** p < 0.01.

Article Snippet: The membranes were blocked with 5% non-fat milk (Thermo Scientific, MA, USA) and probed with rabbit anti-phospho-IFITM3 (dilution 1:2,000, EterLife, China) for 1 h at 37°C.

Techniques: Infection

Journal: Frontiers in Oncology

Article Title: IONPs combined with cytarabine downregulated IFITM3 expression to inhibit acute myeloid leukemia

doi: 10.3389/fonc.2025.1515956

Figure Lengend Snippet: Analyzing the knockdown of IFITM3 efficiency in LV-shIFITM3 KG-1a cells. (A) The efficiency of lentivirus infection of the KG-1a cells was observed under a conventional fluorescent microscope. (B) The IFITM3 mRNA expression in the KG-1a cells was detected by qRT-PCR. (C) The expression of the IFITM3 protein in the KG-1a cells was detected by Western blot. (D) The results from the semi-quantitative analysis of protein expression levels. ****P<0.00001. ns, no statistical significance.

Article Snippet: The membrane was then incubated with the rabbit anti-human GAPDH monoclonal antibody and the rabbit anti-human IFITM3 polyclonal antibody (Wuhan Sanying Biotechnology Co. LTD), respectively, for overnight at 4°C.

Techniques: Knockdown, Infection, Microscopy, Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Oncology

Article Title: IONPs combined with cytarabine downregulated IFITM3 expression to inhibit acute myeloid leukemia

doi: 10.3389/fonc.2025.1515956

Figure Lengend Snippet: Down-regulation of IFITM3 changed the biological properties of the KG-1a cells. (A) The effect of down-regulated IFITM3 on the proliferation ability of KG-1a cells as detected by CCK8 assay. (B) The colony formations of various KG-1a cells detected by soft agar cloning assay (50×); (C) Comparisons of the colony formations; (D) The FCM analysis results of the KG-1a cell cycle changes; (E) Comparisons of the cell cycles; (F) The effects of Ara-C (0.4μM) on the apoptosis of various KG-1a cells analyzed by FCM (24h). (G) The FCM results; (H) The mRNA expression level of caspase 3 in various KG-1a cells as detected by qRT-PCR. *P < 0.05, ***P < 0.001. ns, no statistical significance.

Article Snippet: The membrane was then incubated with the rabbit anti-human GAPDH monoclonal antibody and the rabbit anti-human IFITM3 polyclonal antibody (Wuhan Sanying Biotechnology Co. LTD), respectively, for overnight at 4°C.

Techniques: CCK-8 Assay, Cloning, Expressing, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: IONPs combined with cytarabine downregulated IFITM3 expression to inhibit acute myeloid leukemia

doi: 10.3389/fonc.2025.1515956

Figure Lengend Snippet: The IFITM3 expression and the proliferation of the KG-1a cells. (A) The IFITM3 expression as detected by Western blot after the KG-1a cells were treated with PBS, PBNPs and IONPs, respectively, for 72h; (B) The semi-quantitative analysis of the Western blot results; (C) The IFITM3 expression in the KG-1a cells as detected by Western blot after the cells were treated with PBS, PBNPs and IONPs, respectively, for 72h; (D) The proliferation of the KG-1a cells as detected by the CCK8 assay in the KG-1a cells that were treated with different drugs for 96h; (E) The qRT-PCR-detected expression level of c-myc in the KG-1a cells treated with different drugs for 24h. *P < 0.05, **P < 0.01, ***P < 0.001. ns, no statistical significance.

Article Snippet: The membrane was then incubated with the rabbit anti-human GAPDH monoclonal antibody and the rabbit anti-human IFITM3 polyclonal antibody (Wuhan Sanying Biotechnology Co. LTD), respectively, for overnight at 4°C.

Techniques: Expressing, Western Blot, CCK-8 Assay, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: IONPs combined with cytarabine downregulated IFITM3 expression to inhibit acute myeloid leukemia

doi: 10.3389/fonc.2025.1515956

Figure Lengend Snippet: Impact of IFITM3 knockdown on disease progression in AML model mice. (A) Leukemia cells in peripheral blood and bone marrow; (B) With Reichsen-Giemsa staining; (C) Leukemia cells of bone marrow (left: under a light microscope; right: under a fluorescence microscope) in the AML-bearing mice injected with the Scrambled or the Lv-shIFITM3-KG1a cells; (D) The expression levels of CD33, CD123 and CD11b detected by FCM. (E) Statistical results of the CD33, CD123 and CD11b expression levels. The experiment was repeated twice. *P < 0.05, ***P < 0.001.

Article Snippet: The membrane was then incubated with the rabbit anti-human GAPDH monoclonal antibody and the rabbit anti-human IFITM3 polyclonal antibody (Wuhan Sanying Biotechnology Co. LTD), respectively, for overnight at 4°C.

Techniques: Knockdown, Biomarker Discovery, Staining, Light Microscopy, Fluorescence, Microscopy, Injection, Expressing