Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: WT and Ifitm3 -/- mice were intranasally infected with ( a, b ) 1, 10, or 50 TCID50 of H5N1 avian influenza strain (2 independent experiments for doses 1 and 10 (n=10 mice) and 1 experiment for dose of 50 (n=5 mice)) or with ( c, d ) 1 or 10 TCID50 of H7N3 avian influenza strain (n=5 mice). a , c Viral titers from lung homogenates at day 3 post infection. b, d ELISA quantification of IL-6 levels in lung homogenates at day 3 post infection. a-d Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. Only comparisons between WT and Ifitm3 -/- mice for each dose are shown.

Article Snippet: HeLa IFITM1/2/3 knockout and HAP1 IFITM3 knockout were purchased from ATCC (CRL-3452) and Horizon Discovery Biosciences (HZGHC004186c010), respectively.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: ( a ) Schematic of in vitro infection with potentially zoonotic influenza viruses and representative example from infected A549 human lung cells. ( b ) The indicated A549 cells or THP-1 differentiated macrophages were treated +/- IFNβ for 18 hours, followed by infection with indicated viruses (MOI 1) for 24 hours. Percent infection was determined by flow cytometry and normalized to respective shControl or WT cells without IFNβ pre-treatment. Error bars represent SEM. P values are for the indicated comparisons and were determined by ANOVA followed by Tukey’s multiple comparisons test. Only statistical comparisons between shControl versus shIFITM3 and WT versus IFITM3 -/- are shown. Data are representative of 3 independent experiments each performed in triplicate (n=9). ( c, d ) Western blots of cell lysates at 18 hours +/- IFNβ treatment. Note that commercial IFITM3 antibodies weakly detect IFITM2 in addition to IFITM3. ( e ) The indicated A549 cells were infected with indicated viruses at a range of 0.001 to 10 MOI for 24 hours. Percent infection was determined by flow cytometry. Data are representative of 3 independent experiments each performed in triplicate (n=9). Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. Only comparisons between control and knockdown cells are shown at each dose.

Article Snippet: HeLa IFITM1/2/3 knockout and HAP1 IFITM3 knockout were purchased from ATCC (CRL-3452) and Horizon Discovery Biosciences (HZGHC004186c010), respectively.

Techniques: In Vitro, Infection, Flow Cytometry, Western Blot, Control, Knockdown

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: HAP1 cells were treated +/- IFNβ for 18 hours, followed by infection with indicated viruses (MOI 1) for 24 hours. ( a ) Percent infection was determined by flow cytometry and normalized to results for WT cells without IFNβ pre-treatment. Error bars represent SEM. P values are for the indicated comparisons and were determined by ANOVA followed by Tukey’s multiple comparisons test. Only statistical comparisons between WT versus IFITM3 -/- are shown. Data are representative of 3 independent experiments each performed in triplicate (n=9). ( b ) Western blots of cell lysates at 18 hours +/- IFNβ treatment. ( c ) Example raw flow cytometry data for determination of normalized infection of WT and IFITM3 -/- HAP1 cells +/- IFNb treatment with each of the indicated virus strains as in ( a ). SSC, side scatter; Anti-influenza nucleoprotein antibody was detected in the APC channel with secondary antibody labeled with Alexafluor 647.

Article Snippet: HeLa IFITM1/2/3 knockout and HAP1 IFITM3 knockout were purchased from ATCC (CRL-3452) and Horizon Discovery Biosciences (HZGHC004186c010), respectively.

Techniques: Infection, Flow Cytometry, Western Blot, Virus, Labeling

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: HeLa cells were treated +/- IFNβ for 18 hours, followed by infection with indicated viruses (MOI 1) for 24 hours. ( a ) Percent infection was determined by flow cytometry and normalized to results for WT cells without IFNβ pre-treatment. Error bars represent SEM. P values are for the indicated comparisons and were determined by ANOVA followed by Tukey’s multiple comparisons test. Only statistical comparisons between WT versus IFITM3 -/- are shown. Data are representative of 3 independent experiments each performed in triplicate (n=9). ( b ) Western blots of cell lysates at 18 hours +/- IFNβ treatment. ( c ) Example raw flow cytometry data for determination of normalized infection of WT and IFITM1/2/3 -/- HeLa cells +/- IFNb treatment with each of the indicated virus strains as in a . SSC, side scatter; Anti-influenza nucleoprotein antibody was detected in the APC channel with secondary antibody labeled with Alexafluor 647.

Article Snippet: HeLa IFITM1/2/3 knockout and HAP1 IFITM3 knockout were purchased from ATCC (CRL-3452) and Horizon Discovery Biosciences (HZGHC004186c010), respectively.

Techniques: Infection, Flow Cytometry, Western Blot, Virus, Labeling

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: ( a ) Schematic of mouse passaging experiments. Initial intranasal infections were performed with 1,000 TCID50 of parental viruses. ( b ) Schematic of WT mouse challenge with parental or passaged viruses. ( c-h and j-l ) Groups of WT mice were challenged with equal doses of virus passaged 1, 5, or 10 times through WT or Ifitm3 -/- mice and compared to the parent virus (passage 0). ( c, e ) Viral titers from lung homogenates taken at day 7 ( c represents 2 independent experiments). Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s post hoc test. ( d, f ) ELISA quantification of IL-6 levels in lung homogenates of WT and IFITM3 KO mice at day 7 post infection ( d represents 2 independent experiments). Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. ( g, j ) Viral titers from lung homogenates taken at day 7 ( g ) or day 6 ( j ) post infection. Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s post hoc test. ( h, k ) ELISA quantification of IL-6 levels in lung homogenates of WT and IFITM3 KO mice at day 7 ( h ) or day 6 ( k ) post infection. Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. ( i, m ) Mutations found in the segments of A/California/04/2009 (H1N1) after serial passage through WT or Ifitm3 -/- mice. ( l ) Weight loss. Skull and crossbones indicate humane euthanasia of all animals infected with KO passage 10. Error bars represent SD of the mean, comparisons were made using the Mann-Whitney test (* P = 0.0022, ** P < 0.0001).

Article Snippet: HeLa IFITM1/2/3 knockout and HAP1 IFITM3 knockout were purchased from ATCC (CRL-3452) and Horizon Discovery Biosciences (HZGHC004186c010), respectively.

Techniques: Passaging, Virus, Enzyme-linked Immunosorbent Assay, Infection, MANN-WHITNEY

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: ( a ) Schematic of mouse passaging experiments. Initial intranasal infections were performed with 1,000 TCID50 of parental viruses. ( b ) Schematic of WT mouse challenge with parental or passaged viruses. ( c,d ) Groups of WT mice were challenged with equal doses of virus passaged 1, 5, or 10 times through WT or Stat1 -/- mice and compared to the parent virus (passage 0). ( c ) Viral titers from lung homogenates taken at day 7 post infection. Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. ( d ) ELISA quantification of IL-6 levels in lung homogenates of WT and IFITM3 KO mice at day 7 post infection. Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: HeLa IFITM1/2/3 knockout and HAP1 IFITM3 knockout were purchased from ATCC (CRL-3452) and Horizon Discovery Biosciences (HZGHC004186c010), respectively.

Techniques: Passaging, Virus, Infection, Enzyme-linked Immunosorbent Assay

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: Scientific Reports

Article Title: Combined effects of restriction factors and transduction adjuvants on lentiviral vector gene transfer efficacy

doi: 10.1038/s41598-025-22470-9

Figure Lengend Snippet: RF levels in the overexpression HEK293T cell lines. ( A ). Quantification of RF mRNA expression in the overexpressing cell lines and control cells (Ctl) by qRT-PCR. Because of their very similar sequences (93,3% identity), IFITM2 and IFITM3 transcript levels were measured with the same primer pair. The results correspond to the means and standard deviations of two RNA extractions, each of them measured in duplicates. ( B ). Expression of IFITM proteins in the overexpressing cell lines detected by western blot. IFITM1 (Top) and IFITM2/3 (Bottom) were tested independently in all 6 cell lines and were detected only in the corresponding overexpressing cell lines (apparent MW for both 17 kDa, top panels). Total protein content in each well was checked by looking at actin content (MW 46 kDa, lower panels).

Article Snippet: Membranes were blotted with anti IFITM1 antibody (Proteintech 60,074–1-Ig, 1/5000) or anti IFITM2/3 antibody (Proteintech 66,081–1-Ig, 1/5000).

Techniques: Over Expression, Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: IFITM1 expression determines extracellular vesicle uptake in colorectal cancer

doi: 10.1007/s00018-021-03949-w

Figure Lengend Snippet: Apc mutation induces Ifitm1 expression in intestinal organoids. A Expression analysis of the TCGA dataset in the Oncomine database ( http://www.oncomine.org ) for the indicated probes. B Relative RNA levels of the indicated genes in wild type (WT) and Apc mutant mouse small intestinal organoids (RT-qPCR, n = 4). C Relative RNA levels of Ifitm1 and the Wnt target gene Prox1 in the indicated mouse small intestinal organoids (RT-qPCR, n = 4). D AXIN2, LGR5, MYC, IFITM1 RNA levels in organoids derived from wild type (WT), adenoma and CRC patients (RT-qPCR, n = 2 for adenoma, n = 3 for normal and CRC samples). E Difference in the expression levels between Lgr5 high and Lgr5 low mouse adenoma cells (bioinformatical analysis of the GSE83513 dataset with the GEO2R online tool). F Comparing the expression levels of LGR5, CD44 , CD133 and IFITM1 between LGR5 high and LGR5 low cells in CRC patient-derived organoids (GSE83513 dataset). Unpaired t-test ( A , D ), paired t-test ( B , C ) or t-test with Benjamini and Hochberg false discovery rate ( E , F ) were used with *p < 0.05, **p < 0.01, ***p < 0.005, n.s.: p > 0.05

Article Snippet: Single cells produced from CRC organoids (see above) were suspended at 2 × 10 5 cells/mL in 16.4 μL Nucleofector Solution and 3.6 μl Supplement (Amaxa SF Cell Line 4D-Nucleofector X Kit S, Lonza) in the presence of 1 μg IFITM1 CRISPR/Cas9 KO and 1 μg IFITM1 CRISPR/Cas9 HDR plasmids (Santa-Cruz Biotechnology, sc-416878 and sc-416878-HDR).

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, Derivative Assay

Journal: Cellular and Molecular Life Sciences

Article Title: IFITM1 expression determines extracellular vesicle uptake in colorectal cancer

doi: 10.1007/s00018-021-03949-w

Figure Lengend Snippet: Expression level of IFITM1 in human CRC organoids. A The topology of IFITM1 and binding sites of the N-terminal and C-terminal antibodies. B Flow cytometry with antibodies specific for the N-terminal (N-term) or C-terminal (C-term) parts of IFITM1. Note that samples were either directly labelled or were fixed and permeabilized before labelling. C Whole-mount immunocytochemistry of CRC organoids for IFITM1 with antibodies binding to the N-terminal or C-terminal parts of the protein. Immunostaining was carried out with/without permeabilization (org #1). D , E IFITM1 expression in four CRC organoid lines with the antibody recognizing the C-terminal part of the protein. Representative images ( D ) and the quantification of confocal images ( E ) (whole-mount immunostaining). Scale bars: 50 µm ( C , D )

Article Snippet: Single cells produced from CRC organoids (see above) were suspended at 2 × 10 5 cells/mL in 16.4 μL Nucleofector Solution and 3.6 μl Supplement (Amaxa SF Cell Line 4D-Nucleofector X Kit S, Lonza) in the presence of 1 μg IFITM1 CRISPR/Cas9 KO and 1 μg IFITM1 CRISPR/Cas9 HDR plasmids (Santa-Cruz Biotechnology, sc-416878 and sc-416878-HDR).

Techniques: Expressing, Binding Assay, Flow Cytometry, Immunocytochemistry, Immunostaining

Journal: Cellular and Molecular Life Sciences

Article Title: IFITM1 expression determines extracellular vesicle uptake in colorectal cancer

doi: 10.1007/s00018-021-03949-w

Figure Lengend Snippet: The IFITM1 high CRC cell-derived organoids contain more proliferating cells than IFITM1 low organoids. A The fluorescence sorting strategy for IFITM1 high and IFITM1 low cells and their analysis after sorting with flow cytometry. B IFITM1 protein levels of cells sorted from two CRC organoid lines (capillary-based immunoblotting). Note that actin was used as housekeeping control. C Relative RNA levels of the indicated genes in sorted cells (RT-qPCR, n = 4). D The number of organoids initiated by 20,000 sorted cells on day 7. Each dot represents an individual sorting experiment. E Representative images (left panel) and the diameter of CRC organoids (right panel) derived from IFITM1 low or IFITM1 high sorted cells. F The percentage of KI67+ proliferating cells. Representative images and their quantification from confocal images (whole-mount immunostaining). For E and F , quantifications were carried out from ten images taken from four sorting experiments. Scale bars: 50 µm ( E , F ). Paired t-test ( C ) and Mann–Whitney U-test ( D – F ) were used. ***p < 0.005, n.s.: p > 0.05

Article Snippet: Single cells produced from CRC organoids (see above) were suspended at 2 × 10 5 cells/mL in 16.4 μL Nucleofector Solution and 3.6 μl Supplement (Amaxa SF Cell Line 4D-Nucleofector X Kit S, Lonza) in the presence of 1 μg IFITM1 CRISPR/Cas9 KO and 1 μg IFITM1 CRISPR/Cas9 HDR plasmids (Santa-Cruz Biotechnology, sc-416878 and sc-416878-HDR).

Techniques: Derivative Assay, Fluorescence, Flow Cytometry, Western Blot, Control, Quantitative RT-PCR, Immunostaining, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences

Article Title: IFITM1 expression determines extracellular vesicle uptake in colorectal cancer

doi: 10.1007/s00018-021-03949-w

Figure Lengend Snippet: The IFITM1 high and IFITM1 low CRC cell-derived organoids do not differ in their EV release. A Relative IFITM1 RNA level of sorted cells (d0) and organoids derived from the sorted cells (d7). The RNA levels (normalized to housekeeping) of IFITM1 low samples were taken as 1 in each experiment (RT-qPCR, n = 4). B Immunostaining for IFITM1 directly after sorting or after culturing in 2D or 3D conditions for the indicated time periods (immunostaining and whole-mount immunostaining, confocal microscope images). C , D The percentage of anti-CD63 or anti-CD81-coated positive beads after incubating them in cell-free medium (Ctr), in IFITM1 low or IFITM1 high organoid-derived supernatants and detected by anti-CD63 or anti-CD81 antibody, respectively (flow cytometry). Representative plots ( C ) and quantification of the data ( D ). Note that data were normalized to cell numbers. E , F Nanoparticle Tracking Analysis (NTA) of the organoid supernatants after ultracentrifugation. Representative NTA images ( E ) and data normalized to cell number ( F ). Scale bars: 50 µm ( B ). Paired t-test ( A ) and Mann–Whitney U tests ( D , F ) were used with **p < 0.01 and n.s.: p > 0.05

Article Snippet: Single cells produced from CRC organoids (see above) were suspended at 2 × 10 5 cells/mL in 16.4 μL Nucleofector Solution and 3.6 μl Supplement (Amaxa SF Cell Line 4D-Nucleofector X Kit S, Lonza) in the presence of 1 μg IFITM1 CRISPR/Cas9 KO and 1 μg IFITM1 CRISPR/Cas9 HDR plasmids (Santa-Cruz Biotechnology, sc-416878 and sc-416878-HDR).

Techniques: Derivative Assay, Quantitative RT-PCR, Immunostaining, Microscopy, Flow Cytometry, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences

Article Title: IFITM1 expression determines extracellular vesicle uptake in colorectal cancer

doi: 10.1007/s00018-021-03949-w

Figure Lengend Snippet: IFITM1 high cells have a reduced EV uptake ability. A , B Medium EV (mEV) uptake in IFITM1 low and IFITM1 high sorted cells. Representative images (left panels) and their quantification (right panels). Sorted cells were cultured 2D for 3 days and they were treated with mEVs derived from DiI-labelled HT29 CRC cells ( A ) or human colon fibroblasts ( B ). Note that mEVs show red fluorescence and phalloidin was used to visualize cells. Confocal microscope images were taken from two independent experiments. The shown images are optical slices from the inside of the cells. C Distribution of the DiI signal intensity in IFITM1 low and IFITM1 high cells. Cells were treated with mEVs derived from DiI-labelled fibroblasts. Note the shift of the curve between the two cell populations. D The fluorescent red signal of mEVs in IFITM1 low sorted cells, cultured for 3 days in 2D conditions. mEVs were collected from DiI-labelled fibroblasts and samples were treated with saponin or Triton X-100. Note that the EV signal disappears when using Triton X-100. E The sorting of CRC organoid cells pre-treated with DiI labelled fibroblast-derived small EVs (sEV) overnight in 2D cultures. F Relative RNA levels of IFITM1, AXIN2 and MYC in the sorted EV+ and EV negative CRC cells (RT-qPCR, n = 3). RNA levels (normalized to housekeeping) of the EV+ samples were always taken as 1. G Relative RNA of the indicated genes in CRC organoid cells (cultured in 2D conditions) with or without fibroblast-derived sEVs (RT-qPCR, n = 3). H IFITM1 level and EV uptake of CRC organoid-derived cells (flow cytometry) in the presence/absence of fibroblast-derived mEVs. I The relative percentage of IFITM1 high and IFITM1 low cells within cells with mEV (left panel) or sEV (right panel) in four CRC organoid lines (flow cytometry). J EV-DiI red fluorescent signal intensity in IFITM1 −/low and IFITM1 high cells after treatment with mEVs or sEVs derived from fibroblasts (left panel) or HT29 cells (right panel). Four organoid lines were measured once or twice (flow cytometry). K IFITM1 level and the uptake of liposomes labelled with DiO (representative flow cytometry images). L DiO green fluorescent signal intensity within the DiO-liposome+ population (data were collected from four organoid lines in 4–5 experiments). Scale bars: 20 µm ( A – C ). Paired t-tests ( F , G ), t-test ( C ), Mann–Whitney U-test ( A , B , J , L ) were used with *p < 0.05, ***p < 0.005 and n.d.: p > 0.05

Article Snippet: Single cells produced from CRC organoids (see above) were suspended at 2 × 10 5 cells/mL in 16.4 μL Nucleofector Solution and 3.6 μl Supplement (Amaxa SF Cell Line 4D-Nucleofector X Kit S, Lonza) in the presence of 1 μg IFITM1 CRISPR/Cas9 KO and 1 μg IFITM1 CRISPR/Cas9 HDR plasmids (Santa-Cruz Biotechnology, sc-416878 and sc-416878-HDR).

Techniques: Cell Culture, Derivative Assay, Fluorescence, Microscopy, Quantitative RT-PCR, Flow Cytometry, Liposomes, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences

Article Title: IFITM1 expression determines extracellular vesicle uptake in colorectal cancer

doi: 10.1007/s00018-021-03949-w

Figure Lengend Snippet: Fibroblast-derived EVs induce the proliferation of IFITM1 low CRC cells. A The proportion of IFITM1+ CRC cells in the absence of fibroblast-derived EVs or after adding sEVs or mEVs. B The percentage of IFITM1 low /KI67+ and IFITM1 high /KI67+ cells in CRC organoids after the indicated treatments. C The percentage of KI67+ proliferating cells in IFITM1 high or IFITM1 low CRC cell-derived organoids, treated with fibroblast sEVs or mEVs directly after sorting (whole-mount immunostaining and confocal microscope images). D The proportion of active caspase-3+ apoptotic cells in CRC organoids derived from the indicated cell populations, cultured with or without mEVs or sEVs. Images were taken from three experiments for A , B , C and D . Two-way ANOVA, and Tukey post hoc tests were carried out ( A , B , C , D ) with *p<0.05, **p < 0.01, ***p<0.005 and n.s.: p > 0.05. Note that only the relevant comparisons are shown

Article Snippet: Single cells produced from CRC organoids (see above) were suspended at 2 × 10 5 cells/mL in 16.4 μL Nucleofector Solution and 3.6 μl Supplement (Amaxa SF Cell Line 4D-Nucleofector X Kit S, Lonza) in the presence of 1 μg IFITM1 CRISPR/Cas9 KO and 1 μg IFITM1 CRISPR/Cas9 HDR plasmids (Santa-Cruz Biotechnology, sc-416878 and sc-416878-HDR).

Techniques: Derivative Assay, Immunostaining, Microscopy, Cell Culture

Journal: Cellular and Molecular Life Sciences

Article Title: IFITM1 expression determines extracellular vesicle uptake in colorectal cancer

doi: 10.1007/s00018-021-03949-w

Figure Lengend Snippet: Inactivating IFITM1 results in a higher EV uptake. A Cell surface expression of IFITM1 on CRC organoid cells with (IFITM1 KO ) or without (IFITM1 WT ) CRISPR-based inactivation of the molecule (flow cytometry, Ctr: isotype control from IFITM1 WT cells). Representative histograms (left panel) and their quantification are shown (right panel, n = 4, GeoMean values were analyzed). B Immunostaining of CRC organoids derived from IFITM1 WT or IFITM1 KO cells (confocal microscopic images). C The diameters of IFITM1 WT and IFITM1 KO organoids on day 7 of 3D culturing. D The percentage of CRC cells with labelled EV signal in 2D cultures. Cells were isolated from organoids with the indicated genetic background and they were treated with labelled fibroblast-derived mEVs (evaluation of confocal images from four organoid lines). E The percentage of KI67+ organoid cells in the absence of EVs and in the presence of fibroblast-derived mEVs or sEVs (quantification of confocal microscopic images from four organoid lines). Unpaired t-test ( A ), Mann–Whitney U-test ( C , D ) or Kruskal–Wallis and Dunn tests ( E ) were used. Scale bars: 50 µm ( B ). **p<0.01, ***p<0.005, and n.s.: p>0.05

Article Snippet: Single cells produced from CRC organoids (see above) were suspended at 2 × 10 5 cells/mL in 16.4 μL Nucleofector Solution and 3.6 μl Supplement (Amaxa SF Cell Line 4D-Nucleofector X Kit S, Lonza) in the presence of 1 μg IFITM1 CRISPR/Cas9 KO and 1 μg IFITM1 CRISPR/Cas9 HDR plasmids (Santa-Cruz Biotechnology, sc-416878 and sc-416878-HDR).

Techniques: Expressing, CRISPR, Flow Cytometry, Control, Immunostaining, Derivative Assay, Isolation, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences

Article Title: IFITM1 expression determines extracellular vesicle uptake in colorectal cancer

doi: 10.1007/s00018-021-03949-w

Figure Lengend Snippet: Differential EV uptake by IFITM1 low and IFITM1 high CRC cells has an important effect on the proliferation intensity

Article Snippet: Single cells produced from CRC organoids (see above) were suspended at 2 × 10 5 cells/mL in 16.4 μL Nucleofector Solution and 3.6 μl Supplement (Amaxa SF Cell Line 4D-Nucleofector X Kit S, Lonza) in the presence of 1 μg IFITM1 CRISPR/Cas9 KO and 1 μg IFITM1 CRISPR/Cas9 HDR plasmids (Santa-Cruz Biotechnology, sc-416878 and sc-416878-HDR).

Techniques:

Journal: Virology journal

Article Title: Investigation of antiviral state mediated by interferon-inducible transmembrane protein 1 induced by H9N2 virus and inactivated viral particle in human endothelial cells.

doi: 10.1186/s12985-017-0875-5

Figure Lengend Snippet: Fig. 1 Quantitation of IFITM1 induced by H9N2 virus or inactivated viral particle in HUVECs and BEAS-2Bs. HUVECs and BEAS-2Bs were treated with H9N2 virus (i.e., Virus) or viral particle (i.e., Particle) at MOI of 5. Cells used for RT-PCR and western blot analysis were collected at different time points. a The expression of IFITM1 at mRNA level in HUVECs. b, c The expression of IFITM1 at protein level in HUVECs, and the relative protein level to β-actin. d The expression of IFITM1 at mRNA level in BEAS-2Bs. e, f The expression of IFITM1 at protein level in BEAS-2Bs, and the relative protein level to β-actin. * means particle group and virus group compared with control group (*, P < 0.05. **, P < 0.01, ANOVA). # means particle group compared with H9N2 virus group (#, P < 0.05. ##, P < 0.01, ANOVA)

Article Snippet: IFITM1 specific and control siRNA used in the study were purchased from Santa Cruz Biotechnology, and the siRNA for human cells is a pool of 3 target-specific 19–25 nt siRNAs designed to knock down gene expression.

Techniques: Quantitation Assay, Virus, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Control

Journal: Virology journal

Article Title: Investigation of antiviral state mediated by interferon-inducible transmembrane protein 1 induced by H9N2 virus and inactivated viral particle in human endothelial cells.

doi: 10.1186/s12985-017-0875-5

Figure Lengend Snippet: Fig. 4 Expression of IFITM1 at mRNA level in HUVECs and BEAS-2Bs treated with hemagglutinin (HA) and neuraminidase (NA). HUVECs and BEAS- 2Bs were incubated with HA or NA at concentrations of 0.1, 1 μg/mL. Cells used for RT-PCR analysis were collected at 6, 12, 24 h. a The mRNA levels of IFITM1 induced by HA in HUVECs. b The mRNA levels of IFITM1 induced by NA in HUVECs. c The mRNA levels of IFITM1 induced by HA in BEAS-2Bs. d The mRNA levels of IFITM1 induced by NA in BEAS-2Bs

Article Snippet: IFITM1 specific and control siRNA used in the study were purchased from Santa Cruz Biotechnology, and the siRNA for human cells is a pool of 3 target-specific 19–25 nt siRNAs designed to knock down gene expression.

Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Virology journal

Article Title: Investigation of antiviral state mediated by interferon-inducible transmembrane protein 1 induced by H9N2 virus and inactivated viral particle in human endothelial cells.

doi: 10.1186/s12985-017-0875-5

Figure Lengend Snippet: Fig. 6 Antiviral activity of IFITM1 induced by H9N2 virus infection in HUVECs and BEAS-2Bs. HUVECs and BEAS-2Bs were infected with H9N2 virus at MOI of 5 and incubated for 1 h, then cells were transfected with control siRNA or IFITM1 specific siRNA for 36 h. Virus titer in each group was detected using plaque assay at 36 h postinfection. The effect of IFIMT1 specific siRNA on IFITM1 expression was detected using western blot. a IFITM1 protein level after transfected with siRNA in HUVECs. b IFITM1 protein level after transfected with siRNA in BEAS-2Bs. c Virus titers in HUVECs transfected with control siRNA or IFITM1 specific siRNA. Compared to virus group (control siRNA), the virus titer in virus + siRNA group (IFITM1 specific siRNA) was increased by 13.1 ± 2.4% (P > 0.05, t-test). d Virus titers in BEAS-2Bs transfected with control siRNA or IFITM1 specific siRNA. Compared to virus group (control siRNA), the virus titer in virus + siRNA group (specific siRNA) was increased by 9.7% ± 3.8% (P > 0.05, t-test)

Article Snippet: IFITM1 specific and control siRNA used in the study were purchased from Santa Cruz Biotechnology, and the siRNA for human cells is a pool of 3 target-specific 19–25 nt siRNAs designed to knock down gene expression.

Techniques: Activity Assay, Virus, Infection, Incubation, Transfection, Control, Plaque Assay, Expressing, Western Blot

Journal: Virology journal

Article Title: Investigation of antiviral state mediated by interferon-inducible transmembrane protein 1 induced by H9N2 virus and inactivated viral particle in human endothelial cells.

doi: 10.1186/s12985-017-0875-5

Figure Lengend Snippet: Fig. 7 Antiviral activity of IFITM1 induced by viral particle inoculation in HUVECs and BEAS-2Bs. HUVECs and BEAS-2Bs were inoculated with viral particle at MOI of 5 and incubated for 1 h, then cells were transfected with control siRNA or IFITM1 specific siRNA for 36 h before infected with H9N2 virus at MOI of 5. Virus titer of each group was detected by plaque assay at 36 h postinfection. The effect of IFIMT1 specific siRNA on IFITM1 expression was detected using western blot. a IFITM1 protein level after transfected with siRNA in HUVECs. b IFITM1 protein level after transfected with siRNA in BEAS-2Bs. c Virus titers in HUVECs transfected with control siRNA or IFITM1 specific siRNA. Compared to particle group (control siRNA), the virus titer in particle + siRNA group (IFITM1 specific siRNA) was increased by 60.5 ± 10.7% (P < 0.05, t-test). d Virus titers in BEAS-2Bs transfected with control siRNA or IFITM1 specific siRNA. Compared to particle group (control siRNA), the virus titer in particle + siRNA group (specific siRNA) was increased by 12.9 ± 3.6% (P > 0.05, t-test). * means particle group compared with particle + siRNA group (*, P < 0.05, t-test)

Article Snippet: IFITM1 specific and control siRNA used in the study were purchased from Santa Cruz Biotechnology, and the siRNA for human cells is a pool of 3 target-specific 19–25 nt siRNAs designed to knock down gene expression.

Techniques: Activity Assay, Incubation, Transfection, Control, Infection, Virus, Plaque Assay, Expressing, Western Blot

Journal: Virology journal

Article Title: Investigation of antiviral state mediated by interferon-inducible transmembrane protein 1 induced by H9N2 virus and inactivated viral particle in human endothelial cells.

doi: 10.1186/s12985-017-0875-5

Figure Lengend Snippet: Fig. 8 Overexpression of IFITM1 significantly reduced virus titers in HUVECs and BEAS-2Bs. HUVECs and BEAS-2Bs were transfected with control plasmid (Control) or IFITM1 CRISPR activation plasmid (plasmid) for 36 h, then cells were infected with H9N2 virus at MOI of 5. Virus titer of each group was detected by plaque assay at 36 h postinfection. The overexpression of IFIMT1 was detected by western blot. a IFITM1 protein level after transfected with plasmid in BEAS-2Bs. b IFITM1 protein level after transfected with plasmid in HUVECs. c Virus titers in BEAS-2Bs transfected with control plasmid or IFITM1 CRISPR activation plasmid. Compared to control group (control plasmid), the virus titer in plasmid group (IFITM1 CRISPR activation plasmid) was decreased by 55.72.5 ± 7.53% (P < 0.01, t-test). d Virus titers in HUVECs transfected with control plasmid or IFITM1 CRISPR activation plasmid. Compared to control group (control plasmid), the virus titer in plasmid group (IFITM1 CRISPR activation plasmid) was decreased by 52.76 ± 1.02% (P < 0.01, t-test). * means control group compared with plasmid group (**, P < 0.01, t-test)

Article Snippet: IFITM1 specific and control siRNA used in the study were purchased from Santa Cruz Biotechnology, and the siRNA for human cells is a pool of 3 target-specific 19–25 nt siRNAs designed to knock down gene expression.

Techniques: Over Expression, Virus, Transfection, Control, Plasmid Preparation, CRISPR, Activation Assay, Infection, Plaque Assay, Western Blot

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used at the indicated dilution, in blocking solution: rabbit anti-IFIT1 (1:1000, cat. no. PA3-848, Thermo Fisher Scientific), mouse anti-IFITM1/2/3 (1:500, cat. no. sc-374026, Santa Cruz Biotechnology), mouse anti-COL6A2 (1:500, cat. no. sc-374566, Santa Cruz Biotechnology), mouse anti-COL1A2 (1:500, cat. no. sc-376350, Santa Cruz Biotechnology), mouse anti-TRIM5 (1:500, cat. no. SC-373864, Santa Cruz Biotechnology), mouse anti-HDAC5 (1:500, cat. no. sc-133225, Santa Cruz Biotechnology), mouse anti-HDAC1 (1:500, cat. no. sc-81598, Santa Cruz Biotechnology), mouse anti-FLAG, (1:1000, F3165, Sigma-Aldrich), mouse anti-Myc (1:1000, cat. no. 2276, Cell Signaling Technology), rat anti-α-tubulin (1:10,000, clone YL1/2, cat. no. MCA77G, Serotec), rabbit anti-C6 (1:1000, described in , mouse anti-D8 (1:1000, described in ( )).

Techniques: Virus, Western Blot, Derivative Assay, Subcloning, Recombinant, Protease Inhibitor, Electron Microscopy, Bicinchoninic Acid Protein Assay, TA Cloning, Sequencing, Mass Spectrometry, CRISPR, Disruption, Plasmid Preparation, Software, Quantitative Proteomics, Imaging, Flow Cytometry, Fluorescence, Microscopy

Journal: Antiviral research

Article Title: Spike-mediated viral membrane fusion is inhibited by a specific anti-IFITM2 monoclonal antibody.

doi: 10.1016/j.antiviral.2023.105546

Figure Lengend Snippet: Fig. 1. Design and binding characterization of a specific anti-IFITM2 mAb. A- Analysis of the N-Terminal (NTD) sequences of IFITM2 and IFITM3 and selection of short sequence stretches encompassing the highest number of amino acid substitutions to synthesize oligopeptides for mouse immunization. The sequence alignment and antigen sequences used to immunize the mice are displayed. B- IFITM2 peptide, IFITM3 peptide, an unrelated peptide, recombinant human IFITM1, re combinant human IFITM2 and recombinant human IFITM3 were used in the ELISA test. Results were expressed in O.D. means. C- The monoclonal anti body 5D11B9 was tested for binding to Vero E6 and Calu-3 cell surfaces by flow cytometry. The results showed mAb signal titration from 80 μg/ml to 5 μg/ ml as the mean percentage of positive cells (error bars indicate S.D.). D- Calu-3 cells were seeded on glass coverslips, processed for immunofluorescence and analyzed by confocal imaging. Merged images show DAPI staining of DNA (blue) and IFITM2 (red). Scale bars: 5 μm.

Article Snippet: 96-well microplates were coated overnight at 4 ◦C with peptides (see supplementary information) or proteins: recombinant human IFITM1 (Proteintech, #Ag2320), recombinant human IFITM2 (Proteintech, #Ag17917), recombinant human IFITM3 (Proteintech, Ag17864).

Techniques: Binding Assay, Selection, Sequencing, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Titration, Immunofluorescence, Imaging, Staining

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: Vero (A), 293 (B), and HeLa (C) cells were incubated with the indicated concentrations of IFN-α for 24 h and then transfected with AiV replicon RNA. At the indicated time points after transfection, cell lysates were harvested, and luciferase activity was measured. Each right panel represents the protein levels of IFITM1, IFITM2, or IFITM3 by immunoblotting for cell lysates harvested from experiments shown in the left panels. Data are the means ±SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Incubation, Transfection, Luciferase, Activity Assay, Western Blot

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A–C) 293 (A), HeLa (B), or Vero (C) cell lines stably expressing tetracycline (Tet)-inducible IFITM1, IFITM2 or IFITM3 were cultured with or without Tet for 72 h and then transfected with AiV replicon RNA. Cell lysates were harvested at the indicated time points after transfection and then assayed for luciferase activity. The peak activity obtained for cells in the absence of Tet was taken as 100%. Tet-induced IFITM1, IFITM2, or IFITM3 protein expression in each cell line was detected by Western blotting (bottom panels of (A) and right panel of (B) or right panel of (C)). (D) HeLa cells were transfected with the control, IFITM1, IFITM2, or IFITM3 siRNA for 72 h and then transfected with replicon RNA. At the indicated time points after replicon RNA transfection, cell lysates were harvested and subjected to the luciferase assay. The maximum value obtained for cells treated with control siRNA was taken as 100%. (E) IFITM1, IFITM2, or IFITM3 knockdown was confirmed by Western blotting. (F) Cell viability was determined by the CellTiter-Glo assay. Data are the means ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: The coding regions of IFITM1, IFITM2, and IFITM3 were amplified by PCR using primer pairs containing MluI – EcoRV sites from cDNA clones (OriGene; IFITM1: RC201617, IFITM2: RC202067, IFITM3: RC201635), respectively, and then inserted into the same sites of pACT and pBIND, resulting in pACT-IFITM1, pACT-IFITM2, pACT-IFITM3, pBIND-IFITM1, pBIND-IFITM2, and pBIND-IFITM3.

Techniques: Stable Transfection, Expressing, Cell Culture, Transfection, Luciferase, Activity Assay, Western Blot, Control, Knockdown, Glo Assay