Journal: Intractable & Rare Diseases Research

Article Title: The novel role of IFITM1-3 in myogenic differentiation of C2C12 cells

doi: 10.5582/irdr.2023.01050

Figure Lengend Snippet: Primer sequences for RT-qPCR

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature, and then the membranes were incubated with primary antibodies, namely, rabbit anti-desmin (ab32362, Abcam, USA), rabbit anti-MyoD (ab203383, Abcam, USA), rabbit anti-MYF5 (ab125301, Abcam, USA), rabbit anti-IFITM1 (bs-1031R, Bioss, China), rabbit anti-IFITM2 (bs-15517R, Bioss, China), mouse anti-IFITM3 (bsm-51629M, Bioss, China), and rabbit anti-GAPDH (ab8245, Abcam, USA), at 4°C overnight.

Techniques: Sequencing

Journal: Intractable & Rare Diseases Research

Article Title: The novel role of IFITM1-3 in myogenic differentiation of C2C12 cells

doi: 10.5582/irdr.2023.01050

Figure Lengend Snippet: Increased expression of IFITM1-3 during myogenic differentiation of C2C12 myoblasts. (A) Relative expression of Ifitm1-3 during the myogenic differentiation process. (B) Western blot evaluating the protein levels of IFITM1-3. (C) Quantification of band intensity as described above is shown. The level of proteins was normalized to that of GAPDH. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature, and then the membranes were incubated with primary antibodies, namely, rabbit anti-desmin (ab32362, Abcam, USA), rabbit anti-MyoD (ab203383, Abcam, USA), rabbit anti-MYF5 (ab125301, Abcam, USA), rabbit anti-IFITM1 (bs-1031R, Bioss, China), rabbit anti-IFITM2 (bs-15517R, Bioss, China), mouse anti-IFITM3 (bsm-51629M, Bioss, China), and rabbit anti-GAPDH (ab8245, Abcam, USA), at 4°C overnight.

Techniques: Expressing, Western Blot

Journal: Intractable & Rare Diseases Research

Article Title: The novel role of IFITM1-3 in myogenic differentiation of C2C12 cells

doi: 10.5582/irdr.2023.01050

Figure Lengend Snippet: Knockdown of Ifitm1, 2, and 3 by siRNAs blocks myogenic differentiation in C2C12 cells. (A) Microscopic images of Giemsa staining for C2C12 myoblasts on day 3 of myogenic differentiation after transfection with siRNAs targeting Ifitm1-3. Two different siRNAs were used for each targeting gene. Transfection without siRNAs, but with transfection reagent was set as mock. Magnification: 100×. (B) Percentage fusion on day 3 of myogenic induction and transfection with siRNAs as described above, calculated by dividing the number of nuclei within multinucleated myofibers by the total number of nuclei. NC represents the group without siRNAs and transfection reagents. (C) Downregulated protein expression of myogenin, MyoD, Myf5, and desmin after interference by siRNAs targeting Ifitm1-3. (D) Quantification of band intensity as described above is shown. The level of proteins was normalized to that of GAPDH. Statistical significance: *P< 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature, and then the membranes were incubated with primary antibodies, namely, rabbit anti-desmin (ab32362, Abcam, USA), rabbit anti-MyoD (ab203383, Abcam, USA), rabbit anti-MYF5 (ab125301, Abcam, USA), rabbit anti-IFITM1 (bs-1031R, Bioss, China), rabbit anti-IFITM2 (bs-15517R, Bioss, China), mouse anti-IFITM3 (bsm-51629M, Bioss, China), and rabbit anti-GAPDH (ab8245, Abcam, USA), at 4°C overnight.

Techniques: Staining, Transfection, Expressing

Journal: Intractable & Rare Diseases Research

Article Title: The novel role of IFITM1-3 in myogenic differentiation of C2C12 cells

doi: 10.5582/irdr.2023.01050

Figure Lengend Snippet: Identification and validation of IFITM1,3-interacting proteins. (A) Principle of co-immunoprecipitation and liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). (B) The protein- protein interaction network if IFITM1,3 (overlapped) revealed by STRING analysis. A total of 84 unique homologous proteins are shown in the network. Three clusters are indicated in different colors. Cluster 1: muscle filament sliding (green color); Cluster 2: ribosome series proteins (red color); Cluster 3: regulation of mRNA metabolic process (blue color). Associations are represented by the lines. Thicker lines represent stronger associations. (C) Co-IP assays show the interaction between desmin and IFITM1, 3.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature, and then the membranes were incubated with primary antibodies, namely, rabbit anti-desmin (ab32362, Abcam, USA), rabbit anti-MyoD (ab203383, Abcam, USA), rabbit anti-MYF5 (ab125301, Abcam, USA), rabbit anti-IFITM1 (bs-1031R, Bioss, China), rabbit anti-IFITM2 (bs-15517R, Bioss, China), mouse anti-IFITM3 (bsm-51629M, Bioss, China), and rabbit anti-GAPDH (ab8245, Abcam, USA), at 4°C overnight.

Techniques: Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay

Journal: Intractable & Rare Diseases Research

Article Title: The novel role of IFITM1-3 in myogenic differentiation of C2C12 cells

doi: 10.5582/irdr.2023.01050

Figure Lengend Snippet: Overlapped interacted proteins for IFITM1 and IFITM3

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature, and then the membranes were incubated with primary antibodies, namely, rabbit anti-desmin (ab32362, Abcam, USA), rabbit anti-MyoD (ab203383, Abcam, USA), rabbit anti-MYF5 (ab125301, Abcam, USA), rabbit anti-IFITM1 (bs-1031R, Bioss, China), rabbit anti-IFITM2 (bs-15517R, Bioss, China), mouse anti-IFITM3 (bsm-51629M, Bioss, China), and rabbit anti-GAPDH (ab8245, Abcam, USA), at 4°C overnight.

Techniques:

Journal: Intractable & Rare Diseases Research

Article Title: The novel role of IFITM1-3 in myogenic differentiation of C2C12 cells

doi: 10.5582/irdr.2023.01050

Figure Lengend Snippet: GO and KEGG pathway enrichment analysis of 84 proteins that interact with IFITM1, 3 (overlapped). (A) KEGG classification map of differentially expressed genes. The y-axis shows the metabolic pathway. (B) Biological process (BP). (C) Cellular component (CC). (D) Molecular function (MF). The x-axis represents gene ratio = count/set size. Dot size represents the number of genes, and the color bar represents the Padj-value.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature, and then the membranes were incubated with primary antibodies, namely, rabbit anti-desmin (ab32362, Abcam, USA), rabbit anti-MyoD (ab203383, Abcam, USA), rabbit anti-MYF5 (ab125301, Abcam, USA), rabbit anti-IFITM1 (bs-1031R, Bioss, China), rabbit anti-IFITM2 (bs-15517R, Bioss, China), mouse anti-IFITM3 (bsm-51629M, Bioss, China), and rabbit anti-GAPDH (ab8245, Abcam, USA), at 4°C overnight.

Techniques:

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: IFITMs modulate HCoV-OC43 infection of HepG2 and C3A cells to a similar extent and via the same mechanism. HepG2 and C3A cells were stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing an N-terminally FLAG-tagged IFITM1-EX2 or IFITM3-EX2. The resulting cell lines were infected with HCoV-OC43 at 0.5 MOI. (A) Cells were fixed at 24 hpi and virally infected cells were visualized by IF staining of HCoV-OC43 N protein (green). Cell nuclei were visualized by DAPI staining (blue). (B) HCoV-OC43 NP and exogenously expressed N-terminally FLAG-tagged IFITM proteins and total intracellular IFITM2/3 were determined by Western blotting assays with a monoclonal antibody against the FLAG tag and a rabbit polyclonal antibody against IFITM2/3. β-actin served as a loading control. (C) Intracellular viral RNA was quantified by a qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations ( n = 4). (D) Viral yields were determined with a plaque assay. Error bars indicate standard deviations ( n = 4). (E) HepG2 and C3A stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing IFITM1, IFITM1-EX2, or IFITM3-EX2 were infected with HCoV-OC43pp. Luciferase activities were determined at 72 hpi. Relative infection represents the luciferase activity normalized to that of HepG2 cells transduced with empty vector (pQCXIP). Error bars indicate standard deviations ( n = 6).

Article Snippet: Monoclonal antibody against human IFITM1 (catalog number 60047-1), rabbit polyclonal antibody against human IFITM3 (catalog number 11714-1-AP), which also efficiently recognizes IFITM2 and weakly cross-reacts with IFITM1, were purchased from Proteintech Group, Inc.

Techniques: Infection, Stable Transfection, Transduction, Control, Retroviral, Plasmid Preparation, Expressing, Staining, Western Blot, FLAG-tag, Quantitative RT-PCR, Plaque Assay, Luciferase, Activity Assay

Journal: Scientific reports

Article Title: IFITM proteins inhibit HIV-1 protein synthesis.

doi: 10.1038/s41598-018-32785-5

Figure Lengend Snippet: Figure 7. HIV Nef can help overcome IFITM-mediated restriction of protein synthesis. (A) Level of virus production in HEK293T cells transfected with 0.5 μg expression vectors for IFITMs and 0.5 μg HIV-1 NL4-3 proviral DNA with a deletion (ΔNef) was measured by p24 ELISA 48 hours post-transfection and the data normalized, fold change of virus production compared to vector control is indicated. Differences were assessed with Student’s t tests. (B) Intracellular level of p55/p24 and IFITMs was measured by immunoblotting. (C) SupT1 cells were infected with the indicated dilutions of wild type or Nef-deleted (ΔNef) HIV-1 NL4-3 inoculum and then treated with 1 μg/ml doxycycline to induce IFITM expression post-entry and 5 μM AMD3100 to limit infections to a single round. Level of virus production was measured by p24 ELISA 72 hours post-infection. Differences were assessed with Two-way ANOVA and Bonferroni post-tests. (D) C8166 cells constitutively expressing either vector control or IFITM1 were infected with either wild type or Nef-deleted (ΔNef) HIV-1 NL4-3. Levels of virus production were measured by p24 ELISA at the indicated time-points post-infection and were normalized to the levels of virus produced from vector controls. Differences were assessed with Two-way ANOVA. (E) HEK293T cells were transfected with 0.5 μg expression vectors for IFITMs and 0.5 μg HIV-1 NL4-3 proviral DNA with either wildtype NL4-3 nef or the indicated lentiviral nef alleles. Virus production was measured by p24 ELISA 48 hours post-transfection and the data normalized. Differences were assessed with Student’s t tests. (F) HEK293T cells were transfected with ΔNef HIV-1 NL4-3 proviral DNA, expression vectors for IFITMs and an increasing proportion of HIV-1 Nef-encoding vector versus empty vector in a fixed total quantity of 1 μg. Level of virus production was measured by p24 ELISA 48 hours post- transfection, while levels of viral proteins and IFITM-FLAG expression was analyzed by (G) immunoblotting. Differences were assessed by Student’s t-test. All data show mean + SEM from 3 independent experiments and *denotes p < 0.05.

Article Snippet: The following antibodies were used to detect IFITMs: human IFITM1 (clone 5B5E2, Proteintech), IFITM2 (clone 3D5F7, Proteintech) and human IFITM3 (clone EPR5242, Novus Biologicals).

Techniques: Virus, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Control, Western Blot, Infection, Produced

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Porcine IFITM1 enhances PEDV infection in LLC-PK1 cells. ( A ) Western blot analysis of porcine IFITM1 and IFITM2/3 overexpression in LLC-PK1 cells transduced with lentiviral vectors expressing porcine HA-tagged porcine IFITM1, porcine IFITM2/3, or an empty vector as a negative control. Porcine IFITM1- or IFITM2/3-overexpressing LLC-PK1 cells were infected with the PEDV-HM strain. ( B ) Viral RNA copies in the supernatant were quantified by RT-qPCR and are presented as copy numbers. The data are presented as the means ± s.d. from three technical repeats. ( C ) Viral titers in the supernatant were measured via the TCID 50 assay. The data are presented as the means ± s.d. from three independent experiments. ( D ) LLC-PK1 cells overexpressing porcine IFITM1 or IFITM2/3 were infected with rPEDV-HM-EGFP (0.1 MOI), and the EGFP-fluorescent cells were imaged via fluorescence microscopy at 24 hpi. The green cells represent PEDV-infected cells, while background cells were visualized via white light. Scale bar: 200 µm.

Article Snippet: A monoclonal antibody for human IFITM1 (cat#: 60074-1-lg) was obtained from Proteintech (Shanghai, China).

Techniques: Infection, Western Blot, Over Expression, Transduction, Expressing, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Interaction and colocalization of the PEDV S protein with IFITM proteins. ( A ) Co-IP assays were performed to assess the interaction between the PEDV S1 protein and human or porcine IFITM proteins. HEK293T cells were cotransfected with plasmids encoding PEDV S1-Fc and various HA-tagged IFITM proteins. At 24 h post-transfection, the cells were harvested. Immunoprecipitation was conducted using an Fc tag antibody, and the presence of IFITM proteins was detected by Western blotting (IB: Anti-HA). Whole-cell lysates (WCLs) were analyzed to confirm expression levels (IB: Anti-Fc, Anti-HA, Anti-β-Tubulin). The Co-IP was repeated three times, yielding similar results. ( B ) Confocal microscopy images showing the intracellular localization of porcine IFITM1 in LLC-PK1 cells are presented. IFITM1 (red) colocalizes with clathrin (green), EEA1 (green), Rab7 (green), and LAMP7 (green), as indicated in the merged images. DAPI (blue) was used to stain the nuclei. ( C ) Confocal microscopy images demonstrating the colocalization of the PEDV S protein (red) with HA-IFITM1 (green) in LLC-PK1 cells are shown. The merged image shows the nuclei stained with DAPI (blue).

Article Snippet: A monoclonal antibody for human IFITM1 (cat#: 60074-1-lg) was obtained from Proteintech (Shanghai, China).

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Expressing, Confocal Microscopy, Staining

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Porcine IFITM1 enhanced PEDV infection in porcine small intestinal organoids. ( A ) Porcine small intestinal crypts were isolated from neonatal piglets and cultured in vitro . A representative image depicting the morphology of porcine small intestinal organoids is shown. Scale bar: 200 µm. ( B ) Immunofluorescence confirmation of HA-tagged porcine IFITM1 overexpression in PSIOs. Representative images displaying IFITM1 (HA, green) and nuclei (DAPI, blue) are presented. ( C ) The expression of IFITM1 was verified via immunofluorescence in a 2D organoid culture system. Representative images showing HA (green) and nuclei (DAPI, blue) staining are presented. ( D ) Analysis of viral infection in IFITM1-overexpressing and control 2D organoids following PEDV infection is shown. IFITM1-overexpressing or control 2D organoids were infected with PEDV-HM (0.1 MOI). At 24 hpi, the virus titer in the supernatant was quantified according to the TCID 50 . The data are presented as the means ± s.d. from three independent experiments. **, P < 0.01. ( E ) IFITM1-overexpressing or control 2D organoids were infected with rPEDV-HM-EGFP (0.1 MOI), and the EGFP-fluorescent cells were imaged via fluorescence microscopy at 24 hpi. The green cells represent PEDV-infected cells, and the background cells were visualized under white light. Scale bar: 200 µm.

Article Snippet: A monoclonal antibody for human IFITM1 (cat#: 60074-1-lg) was obtained from Proteintech (Shanghai, China).

Techniques: Infection, Isolation, Cell Culture, In Vitro, Immunofluorescence, Over Expression, Expressing, Staining, Control, Virus, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: In Vivo Functional Requirement of the Mouse Ifitm1 Gene for Germ Cell Development, Interferon Mediated Immune Response and Somitogenesis

doi: 10.1371/journal.pone.0044609

Figure Lengend Snippet: ( A ) Shows the scheme of the linearized targeting vector used for electroporation in mouse ES cells. The coding sequence of the lacZ reporter gene (lacZ) was inserted in frame into the first codon of the Ifitm1 gene. The three Ifitm1 exons are indicated as I, II, and III. The lacZ gene is followed by a poly-adenylation signal (bGHpA) and a neomycin/kanamycin selection cassette (neo/kan) that was flanked by loxP sites (L) for subsequent deletion of the cassette. The backbone of the vector (vb) included the coding sequence for the diphtheria toxin fragment A (DTA) to support the selection of clones carrying the homologous recombination of the targeting vector. The location of the probe used for Southern blotting (sp) and PvuII restriction sites (P) are also indicated. The PvuII restriction sites that are relevant for the diagnostic restriction fragments in Southern blots in combination with DNA probe sp are marked with an underline ( P ). ( B ) Shows a scheme of the wildtype Ifitm1 locus on mouse chromosome 7. The three Ifitm1 exons (I, II, and III) are indicated as boxes. Black shading in the boxes indicates the coding sequence of Ifitm1 . The genomic region that was amplified by PCR to genotype the Ifitm1 wt allele is indicated (wt-PCR). ( C ) Shows the Ifitm1 locus following homologous recombination in mouse ES cells. As result of recombination, the complete Ifitm1 coding sequence is replaced by the lacZ reporter gene and the selection cassette (lacZ-bGHpA-Lneo/kanL). The genomic region that is covered by the homologous sequence of the targeting vector (tv) is designated. The genomic region that was amplified by PCR to genotype the targeted Ifitm locus in the electroporated ES cells following G418 selection is indicated (ES-PCR). ( D ) Shows a scheme of the Ifitm1 tm1IEG loss-of-function allele that was generated by expression of the Cre recombinase and subsequent deletion of the neo/kan selection cassette from the targeted Ifitm1 allele shown in ( C ). As result of this excision, a single loxP site (L) is left behind as indicated. The genomic region that was amplified by PCR to genotype the targeted Ifitm tm1IEG loss-of-function allele in mice obtained from matings between chimeric mice and Cre expressing mice is indicated (flox-ko-PCR). The size bar (bottom left) indicates 1 kb length.

Article Snippet: Primary antibodies for IHC were human Ifitm1 (Sigma-Aldrich, HPA004810) and human CK7 (DAKO, M7018).

Techniques: Plasmid Preparation, Electroporation, Sequencing, Selection, Clone Assay, Homologous Recombination, Southern Blot, Diagnostic Assay, Amplification, Generated, Expressing

Journal: PLoS ONE

Article Title: In Vivo Functional Requirement of the Mouse Ifitm1 Gene for Germ Cell Development, Interferon Mediated Immune Response and Somitogenesis

doi: 10.1371/journal.pone.0044609

Figure Lengend Snippet: Most significantly over-represented Gene Ontology (GO) terms among regulated genes in the comparative transcriptome analysis of Ifitm1 tm1IEG/tm1IEG versus Ifitm1 wt/wt lungs and list of regulated genes annotated with the respective GO terms.

Article Snippet: Primary antibodies for IHC were human Ifitm1 (Sigma-Aldrich, HPA004810) and human CK7 (DAKO, M7018).

Techniques: Activation Assay, Chemotaxis Assay

Journal: PLoS ONE

Article Title: In Vivo Functional Requirement of the Mouse Ifitm1 Gene for Germ Cell Development, Interferon Mediated Immune Response and Somitogenesis

doi: 10.1371/journal.pone.0044609

Figure Lengend Snippet: ( A and B ) Detection of Ifitm1 expression in adult mouse lung by whole organ X-Gal staining. LacZ expression was detected in the bronchia of the lung of Ifitm1 tm1IEG/wt adult mice ( A ) but not in wildtype lungs ( B ). ( C and D ) Subsequent Histological sections of stained Ifitm1 tm1IEG/wt mouse lungs revealed lacZ expression in the cells of the bronchial epithelium of Ifitm1 tm1IEG/wt mice. ( E ) By immunohistochemistry on human adult lung tissue, IFITM1 expression was detected in the columnar- and in the basal cells of the bronchial epithelium. ( F ) Immunohistochemical detection of CYTOKERATIN -7 (CK7) was used to identify columnar cells of the bronchial epithelium. Scale bars indicate 100 µm.

Article Snippet: Primary antibodies for IHC were human Ifitm1 (Sigma-Aldrich, HPA004810) and human CK7 (DAKO, M7018).

Techniques: Expressing, Staining, Immunohistochemistry, Immunohistochemical staining

Journal: PLoS ONE

Article Title: In Vivo Functional Requirement of the Mouse Ifitm1 Gene for Germ Cell Development, Interferon Mediated Immune Response and Somitogenesis

doi: 10.1371/journal.pone.0044609

Figure Lengend Snippet: ( A and B ) Whole organ X-Gal staining of the thymus of heterozygous knockout animals exhibited lacZ expression whereas thymus of wildtype littermate mice did not stain for X-Gal. ( C and D ) Histological sections of stained thymus revealed cells of the medulla expressing the lacZ reporter gene in Ifitm1 tm1IEG/wt mice ( C ) but not in thymus from wildtype littermate mice ( D ). Scale bars indicate 100 µm.

Article Snippet: Primary antibodies for IHC were human Ifitm1 (Sigma-Aldrich, HPA004810) and human CK7 (DAKO, M7018).

Techniques: Staining, Knock-Out, Expressing

Journal: PLoS ONE

Article Title: In Vivo Functional Requirement of the Mouse Ifitm1 Gene for Germ Cell Development, Interferon Mediated Immune Response and Somitogenesis

doi: 10.1371/journal.pone.0044609

Figure Lengend Snippet: ( A and D ) IFITM1 is expressed in non-neoplastic alveolar and bronchial epithelia. ( B and E ) The IFITM1 gene is highly overexpressed in squamous cell carcinomas of the human lung and ( C and F ) in adenocarcinomas of the human lung.

Article Snippet: Primary antibodies for IHC were human Ifitm1 (Sigma-Aldrich, HPA004810) and human CK7 (DAKO, M7018).

Techniques: