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: iScience

Article Title: IFITM1 inhibits trophoblast invasion and is induced in placentas associated with IFN-mediated pregnancy diseases

doi: 10.1016/j.isci.2023.107147

Figure Lengend Snippet: Upregulation of IFITMs in HTR8/SVneo cells following IFN-β treatment was evaluated by western blot and immunofluorescence IFN-β inhibits invasion of HTR8/SVneo cells (A–E) and primary human EVCTs (F–I). HTR8/SVneo cells and EVCTs were incubated for 48 h with IFN-β (10, 100, or 1000 IU/mL). (A) Cell lysates (40 μg) were loaded in 4–15% SDS-PAGE gradient gels and blots were probed for IFITM1, IFITM2/3, and vinculin expression. One representative experiment (out of 3) is shown. (B) Representative image of HTR8/SVneo cells immunostained for cytokeratin 7 (marker of trophoblast, magenta) and IFITM1 (green) or IFITM2/3 (red), and then counterstained with DAPI (blue). Scale bars: 10 μm. Inhibition of HTR8/SVneo cell invasion following IFN-β treatment , quantified using IncuCyte technology (C–E) . HTR8/SVneo cells were cultured in a monolayer in 96-well plates. Cells were treated for 48 h, then a wound was made using a 96-well wound maker; this was covered by Matrigel and cells were imaged with Incucyte every 30 min for 48 h. (C) Representative images of HTR8/SVneo cell invasion at 48 h. The blue region denotes the area of the initial wound (light blue line) covered by advancing cells. (D) Time course of wound closure expressed as relative wound density (%). Values are reported as mean ± SEM. (E) Comparison between vehicle and IFN-β-treated cells was performed using an analysis of the area under the curve of the replicates represented in (D). Values are reported as mean + SEM (n = 6 wells/condition, n = 3 experiments). Statistical analysis was performed with one-way ANOVA to compare treatment to vehicle controls. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Upregulation of IFITMs in EVCTs following IFN-β treatment was evaluated by western blot (F) and immunofluorescence (G) . (F) Cell lysates (40 μg) from isolated EVCTs were loaded in 4–15% SDS-PAGE gradient gels and blots were probed for IFITM1, IFITM2/3, and vinculin expression. One representative experiment (out of 3) is shown. (G) Representative image of isolated EVCTs and placental explants immunostained for cytokeratin 7 (marker of trophoblast, magenta) and IFITM1 (green) or IFITM2/3 (red), and then counterstained with DAPI (blue). Scale bars: 10 μm. Inhibition of EVCT invasion following IFN-β treatment using villous explant culture model (H–I) . (H) Representative image of extravillous explants cultured on Matrigel and immunostained for integrin α5 (ITGA5, marker of invasion, red). Scale bars: 100 μm. (I) Statistical analysis of the invasion distance of ITGA5 + EVCTs, as representatively shown in H. Values are presented as mean + SD (n = 3 explants per condition, n = 3 placentas). One-way ANOVA was performed to compare treatment to vehicle control; ∗∗∗∗p < 0.0001.

Article Snippet: Rabbit anti-IFITM1 polyclonal antibody , Sigma-Aldrich , Cat# HPA004810; RRID: AB_1851419.

Techniques: Western Blot, Immunofluorescence, Incubation, SDS Page, Expressing, Marker, Inhibition, Cell Culture, Isolation

Journal: iScience

Article Title: IFITM1 inhibits trophoblast invasion and is induced in placentas associated with IFN-mediated pregnancy diseases

doi: 10.1016/j.isci.2023.107147

Figure Lengend Snippet: Ectopic expression of FLAG and IFITMs in HTR8/SVneo cells transduced with IFITM1–3 was verified by western blot, immunofluorescence, and flow cytometry IFITM1 inhibits invasion of HTR8/SVneo cells (A–E) and primary human EVCTs. (A) Cell lysates (40 μg) were loaded in 4–15% SDS-PAGE gradient gels and blots were probed for IFITM1, IFITM2/3, and vinculin expression. A representative experiment is shown. (B) Representative image of transduced HTR8/SVneo cells immunostained for FLAG (yellow) and IFITM1 (green) or IFITM2/3 (red), and then counterstained with DAPI (blue). Scale bar: 10 μm. (C) HTR8/SVneo cells transduced with a vector containing IFITM1 (green), IFITM2 (orange), IFITM3 (magenta), or a control empty vector (dark blue) were stained with antibodies for IFITM1, IFITM2/3, and FLAG, as well as corresponding isotypes. Cells were then analyzed by flow cytometry. Inhibition of IFITM1-transduced HTR8/SVneo cell invasion , quantified using IncuCyte technology (D–F) . HTR8/SVneo cells transduced with IFITM1–3 were cultured in a monolayer in 96-well plates and a wound was made using a 96-well wound maker; this was covered by Matrigel and cells were imaged with Incucyte every 30 min for 48 h. (D) Representative images of transduced HTR8/SVneo cell invasion at 48 h. The blue region denotes the area of the initial wound (light blue line) covered by advancing cells. (E) Time course of wound closure expressed as relative wound density (%). Values are reported as mean ± SEM of the percentage. (F) Comparison between cells transduced with IFITM1–3 and the empty vector was performed using an analysis of the area under the curve of the replicates represented in (E). Values are represented as mean + SEM (n = 6 wells/condition, n = 3 experiments). Statistical analysis was performed with one-way ANOVA to compare treatment to vehicle controls. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Ectopic expression of GFP , FLAG , and IFITMs in EVCT explants transduced with IFITM1–3 was verified by immunofluorescence (G–J) . (G) Representative image of placental explants transduced with GFP or IFITM1–3, cultured on Matrigel, and immunostained for FLAG (yellow), GFP (green), and IFITM1 (red) or IFITM2/3 (magenta), and then counterstained with DAPI (blue). Scale bars: 100 μm. (H) Representative image of EVCT explants transduced with GFP or IFITM1–3, cultured on Matrigel, and immunostained for FLAG (yellow), GFP (green), and ITGA5 (red) or HLAG (magenta), and then counterstained with DAPI (blue). Scale bars: 100 μm. Inhibition of IFITM1-transduced EVCT invasion using villous explant culture model (I–J) . (I) Representative image of transduced extravillous explants cultured on Matrigel and immunostained for integrin α5 (ITGA5, marker of invasion, red). Scale bars: 100 μm. (J) Statistical analysis of the invasion distance of ITGA5 + EVCTs, as representatively shown in I. Values are presented as mean + SD (n = 3 explants per condition, n = 3 placentas). One-way ANOVA was performed to compare treatment to pTRIP-GFP control; ∗∗∗∗p < 0.0001.

Article Snippet: Rabbit anti-IFITM1 polyclonal antibody , Sigma-Aldrich , Cat# HPA004810; RRID: AB_1851419.

Techniques: Expressing, Transduction, Western Blot, Immunofluorescence, Flow Cytometry, SDS Page, Plasmid Preparation, Staining, Inhibition, Cell Culture, Marker

Journal: iScience

Article Title: IFITM1 inhibits trophoblast invasion and is induced in placentas associated with IFN-mediated pregnancy diseases

doi: 10.1016/j.isci.2023.107147

Figure Lengend Snippet: Induction of IFITM1 expression in placental tissues from IFN-induced pathologies of pregnancy (A) Representative images of CK7, IFITM1, and HLAG immunostaining and HES staining in placental tissues from CMV- and bacterial-infected patients (left panel) and non-infected controls (right panel). Yellow asterisks represent EVCTs (CK7+, HLGA+). Scale bars: 200 μm. The insets depict a higher magnification of EVCT labeling. Scale bar: 20 μm. (B) Quantification of IFITM1 protein expression in EVCTs from CMV- and bacterial-infected and non-infected control pregnancies at three different weeks of gestation (19, 24, and 34 WG, n = 1 per condition, 25 non-overlapping ROIs per placenta). (C) Statistical analyses of IFITM1 protein expression of data shown in B combined by all gestational ages per (n = 3 placentas (19, 24, and 34 WG) per condition (CMV- or bacterial-infected and non-infected), 75 non-overlapping ROIs per condition). Values are presented as mean + SD. One-way ANOVA was performed to compare infected to non-infected controls; ∗∗∗∗p < 0.0001.

Article Snippet: Rabbit anti-IFITM1 polyclonal antibody , Sigma-Aldrich , Cat# HPA004810; RRID: AB_1851419.

Techniques: Expressing, Immunostaining, Staining, Infection, Labeling

Journal: iScience

Article Title: IFITM1 inhibits trophoblast invasion and is induced in placentas associated with IFN-mediated pregnancy diseases

doi: 10.1016/j.isci.2023.107147

Figure Lengend Snippet:

Article Snippet: Rabbit anti-IFITM1 polyclonal antibody , Sigma-Aldrich , Cat# HPA004810; RRID: AB_1851419.

Techniques: Recombinant, Electron Microscopy, Selection, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Software