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: BMC Genomics

Article Title: Annexin A2 (ANXA2) regulates the transcription and alternative splicing of inflammatory genes in renal tubular epithelial cells

doi: 10.1186/s12864-022-08748-6

Figure Lengend Snippet: ANXA2 regulated inflammatory gene mRNA and protein expression in HK2 cells. A Top ten GO biological processes terms enriched by upregulated DEGs in shANXA2 cells vs shCtrl cells. B Top ten KEGG functional pathways enriched by upregulated DEGs in shANXA2 cells vs shCtrl cells. C Top ten KEGG functional pathways enriched by downregulated DEGs in shANXA2 cells vs shCtrl cells. D Validation of mRNA expression of CCL5, IFI6, IFI44, IFITM1,and LTB by qRT-PCR assay. E Validation of mRNA expression of IRF7 and ISG15 by qRT-PCR assay. F Representative images showing protein levels of ANXA2, CCL5, IFI6, IFI44, IFITM1, LTB, IRF7 and ISG15 in LV-shANXA2 group vs LV-shCtrl group. Results are represented as mean ± SD.( n = 3,* P < 0.05, ** P < 0.01, *** P < 0.001, calculated using the student’s t-test)

Article Snippet: Anti-rabbit secondary antibody conjugated to horseradish peroxidase (Boster, BA1054, Wuhan, China) was incubated with the membranes for 1 h. Primary antibodies anti-ANXA2(AF 5420), anti-CCL5 (DF5151), anti-IFI6 (DF10115), anti-IFITM1 (DF 2513), anti-LTB (DF 3243), anti-IRF7 (DF7503), and ISG15 (DF6316) were from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Expressing, Functional Assay, Quantitative RT-PCR

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: 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: Oncogene

Article Title: IFITM1 plays an essential role in the antiproliferative action of interferon-gamma.

doi: 10.1038/sj.onc.1209807

Figure Lengend Snippet: Figure 1 IFITM1 mediates the antiproliferative action of IFN-g. (a) Effect of IFN-g on the proliferation of BEL-7404 and Chang liver cells. BEL-7404 (left panel) and Chang liver (right panel) cells were treated with IFN-g (500 U/ml) for the indicated times, followed by measuring cell viability. Data are expressed relative to the cell viability detected at the initial time point, and are shown as mean7s.d. of three independent experiments. (b) RNAi effect in stable RNAi cells derived from BEL-7404 (7RI-1 and 7RI-2) and Chang liver (CRI-1 and CRI-2). As controls, 7RS and CRS were GFP-RNAi vector-transfected cells. (c) IFITM1 RNAi and control cells were treated with the indicated concentrations of IFN-g for 24 h, followed by measuring IFITM1 mRNA using quantitative real-time RT–PCR. Data normalized to b-actin mRNA are expressed relative to that of untreated control cells, and are shown as mean7s.d. of three independent experiments. (d) Effect of IFN-g on the proliferation of IFITM1 RNAi cells. BEL-7404 and Chang liver IFITM1 RNAi and control cells were treated with the indicated concentrations of IFN-g for 96 h. Data are expressed relative to the untreated controls, and shown as mean7s.d. of three independent experiments.

Article Snippet: Immunoblotting was performed as described previously (Huang et al., 2002) with primary antibodies against p21, ERK, phosphor-ERK, phospho-p53 (Thr55), actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (Upstate Biotechnology, Lake Placid, NY, USA), myc (Oncogene Science, Uniondale, NY, USA), or IFITM1 (Boster, Wuhan, China).

Techniques: Derivative Assay, Plasmid Preparation, Transfection, Control, Quantitative RT-PCR

Journal: Oncogene

Article Title: IFITM1 plays an essential role in the antiproliferative action of interferon-gamma.

doi: 10.1038/sj.onc.1209807

Figure Lengend Snippet: Figure 2 IFITM1 is a negative regulator of cell proliferation and tumorigenesis. (a) Effect of IFITM1 overexpression on the proliferation of BEL-7404 (left panel) and QSG-7701 (right panel) cells. Cells were transfected with pcDNA-IFITM1 (IFITM1), pcDNA (Vector), EGFP-N1 (GFP) and IFITM1-GFP (IFITM1-G) for 48 h, followed by measuring cell viability. Data are expressed relative to the viability of cells transfected with pcDNA vector, and are shown as mean7s.d. of three independent experiments. Asterisks indicate statistically significant difference (*Po0.05, **Po0.01). (b) Growth rates of Chang liver IFITM RNAi, control and parental (Par) cells. Data are mean7s.d. of three independent experiments. (c) The weight of tumors from the nude mice that received injections of CRI-1, CRI-2 and CRS cells (bars, s.d.). (d) Quantitative real-time RT–PCR measurement of IFITM1 mRNA in clinical HCC samples. Data are mean7s.d. of three independent experiments.

Article Snippet: Immunoblotting was performed as described previously (Huang et al., 2002) with primary antibodies against p21, ERK, phosphor-ERK, phospho-p53 (Thr55), actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (Upstate Biotechnology, Lake Placid, NY, USA), myc (Oncogene Science, Uniondale, NY, USA), or IFITM1 (Boster, Wuhan, China).

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Quantitative RT-PCR

Journal: Oncogene

Article Title: IFITM1 plays an essential role in the antiproliferative action of interferon-gamma.

doi: 10.1038/sj.onc.1209807

Figure Lengend Snippet: Figure 3 IFITM1 inhibits activity of ERK. (a) Effect of IFITM1 overexpression on phosphorylation of ERK. BEL-7404 cells were transfected with increasing amounts of IFITM1-myc for 36 h. Lysates were blotted with phospho-ERK and myc antibodies, and reprobed with total ERK antibody. (b) Effect of IFITM1 overexpression on ERK-mediated transcription. BEL-7404 cells were transfected with increasing amounts of IFITM1-myc in the presence of ERK2, ERK luciferase reporter plasmids and pRL-TK. Data are shown as mean7s.d. of three independent experiments. *Po0.05, **Po0.01 as compared to vector-transfected cells. (c) Detection of phospho-ERK in Chang liver IFITM1 RNAi and control cells. Total ERK was shown as a loading control. (d and e) Effect of IFN-g on phosphorylation of ERK in BEL-7404 parental (d) and IFITM1 RNAi (e) cells. Cells were treated with ( þ ) or without () IFN-g (500 U/ml) and incubated for the indicated times. Lysates were blotted with phosphor-ERK and IFITM1 antibody, and reprobed with ERK antibody as a loading control.

Article Snippet: Immunoblotting was performed as described previously (Huang et al., 2002) with primary antibodies against p21, ERK, phosphor-ERK, phospho-p53 (Thr55), actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (Upstate Biotechnology, Lake Placid, NY, USA), myc (Oncogene Science, Uniondale, NY, USA), or IFITM1 (Boster, Wuhan, China).

Techniques: Activity Assay, Over Expression, Phospho-proteomics, Transfection, Luciferase, Plasmid Preparation, Control, Incubation

Journal: Oncogene

Article Title: IFITM1 plays an essential role in the antiproliferative action of interferon-gamma.

doi: 10.1038/sj.onc.1209807

Figure Lengend Snippet: Figure 4 IFITM1 arrests cell growth in the G1 phase in a p53-dependent manner. (a) Effect of IFITM1 overexpression on the cell cycle progression in the indicated cell lines. BEL-7404, QSG-7701, HeLa and Saos-2 cells were transfected with plasmid expressing GFP alone (GFP) or IFITM1-GFP fusion protein (IFITM1-GFP). After 48 h, cells were subjected to FACS analysis and separated into GFP-negative () and GFP-positive ( þ ) populations. Results shown represent three independent experiments. (b) RNAi effect in BEL-7404 p53 RNAi cells. Lysates of p53 RNAi (7RP-1 and 7RP-2), control (7RS) and parental (Par) cells were blotted with anti-p53 antibody. Actin was shown as a loading control. (c) Effect of IFITM1 overexpression on the cell cycle progression in p53 RNAi cells. BEL-7404 control and p53 RNAi cells were transfected with GFP (G) or IFITM1-GFP (I). After 48 h, GFP-positive populations were analysed for cell cycle distributions. Data represent three independent experiments.

Article Snippet: Immunoblotting was performed as described previously (Huang et al., 2002) with primary antibodies against p21, ERK, phosphor-ERK, phospho-p53 (Thr55), actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (Upstate Biotechnology, Lake Placid, NY, USA), myc (Oncogene Science, Uniondale, NY, USA), or IFITM1 (Boster, Wuhan, China).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Control

Journal: Oncogene

Article Title: IFITM1 plays an essential role in the antiproliferative action of interferon-gamma.

doi: 10.1038/sj.onc.1209807

Figure Lengend Snippet: Figure 5 IFITM1 enhances the transcriptional activity of p53. (a) Effect of IFITM1 overexpression on p53 transcriptional activity. Saos-2 (left panel) and BEL-7404 (right panel) cells were transfected with plasmids as indicated in the presence of p53 reporter plasmids. Data are shown as mean7s.d. of three independent experiments. *Po0.05, **Po0.01. (b and c) Effect of IFITM1 overexpression on the protein (b) and mRNA (c) levels of p53 and p21. BEL-7404 cells were transfected with increasing amounts of IFITM1-myc for 36 h. (b) Lysates were blotted with p53, p21, myc and actin antibodies. (c) Expression level of p21 and p53 was determined using quantitative real-time RT–PCR.

Article Snippet: Immunoblotting was performed as described previously (Huang et al., 2002) with primary antibodies against p21, ERK, phosphor-ERK, phospho-p53 (Thr55), actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (Upstate Biotechnology, Lake Placid, NY, USA), myc (Oncogene Science, Uniondale, NY, USA), or IFITM1 (Boster, Wuhan, China).

Techniques: Activity Assay, Over Expression, Transfection, Expressing, Quantitative RT-PCR

Journal: Oncogene

Article Title: IFITM1 plays an essential role in the antiproliferative action of interferon-gamma.

doi: 10.1038/sj.onc.1209807

Figure Lengend Snippet: Figure 6 IFITM1 stabilizes p53 protein by inhibiting Thr55 phosphorylation on p53. (a) Effect of IFITM1 on degradation of endogenous p53 protein. Twenty-four hours after transfection with IFITM1-myc or empty vector, BEL-7404 cells were treated with CHX (10 mg/ml) for the indicated times. Quantitative analysis of p53 protein was illustrated in the right panel. Data, normalized to actin, are shown as mean7s.d. of four independent experiments. *Po0.05, **Po0.01. (b) BEL-7404 cells were co-transfected with p53 and increasing amounts of IFITM1 for 36 h. Lysates were immunoblotted with p-p53 (Thr55), total p53, p-ERK, total ERK, p21 and myc antibodies. Actin was shown as a loading control. (c) Effect of IFITM1 on degradation of ectopic p53 wild-type and mutant T55A protein. After 24 h of transfection, Saos-2 cells were treated with CHX (10 mg/ml) for the indicated times. Lysates were blotted with p53 antibody. Actin was shown as a loading control. Quantitative analysis of p53-wild type and mutant T55A protein was illustrated in the right panel. Data, normalized to actin, are shown as mean7s.d. of three independent experiments.

Article Snippet: Immunoblotting was performed as described previously (Huang et al., 2002) with primary antibodies against p21, ERK, phosphor-ERK, phospho-p53 (Thr55), actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (Upstate Biotechnology, Lake Placid, NY, USA), myc (Oncogene Science, Uniondale, NY, USA), or IFITM1 (Boster, Wuhan, China).

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Control, Mutagenesis