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: 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: 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

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Δ20 IFITM2 differentially restricts X4 and R5 HIV-1

doi: 10.1073/pnas.1619640114

Figure Lengend Snippet: Characterization of expression of Δ20 IFITM2. (A) Schematic representation of amino acid sequences of IFITM1, IFITM2, IFITM3, and Δ20 IFITM2. Identical amino acid sequences among different IFITM proteins are colored in red. Immunogens of commercial antibodies used in our studies are shown. (B) Vector-transduced GHOST R5 cells or GHOST R5 cells expressing FLAG-tagged FL IFITM2, Δ20 IFITM2, or IFITM3 were analyzed by Western blotting using the indicated antibodies. Note that the anti-IFITM2/3/Δ20 antibody recognizes FL IFITM2, Δ20 IFITM2, and IFITM3. (C) Experiments were similar to the experiments in Fig. 1E except that Δ20 IFITM2 expression in anti-CD3 and anti-CD28 antibody-activated CD4+ T cells from three different donors was analyzed. (D) Vector-transduced and Δ20 IFITM2-expressing Jurkat E6-1 R5 cells were labeled with anti-IFITM2/3/Δ20 primary and Alexa 488-conjugated secondary antibodies. Cells were then analyzed by flow cytometry. The histogram images of the Alexa 488 signal in the indicated cells and in cells labeled with a secondary antibody alone are shown. (E) Experiments were similar to the experiments in D except that anti-CD3 and anti-CD28 antibody-activated CD4+ T cells were analyzed. Experiments were similar to the experiments in C except that Δ20 IFITM2 expression in monocytes (F) and moDCs (G) was analyzed. Arrows indicate Δ20 IFITM2 bands. Note that the upper bands detected by the anti-IFITM2/3/Δ20 antibody could be FL IFITM2 or IFITM3. (H) Experiments were similar to the experiments in C except that anti-CD3 and anti-CD28 antibody-activated CD4+ T cells were treated with media, 1,000 U/mL IFN-β, or 1 μg/mL PHA for 2 d.

Article Snippet: Note that the upper bands detected by the anti-IFITM2/3/Δ20 antibody could be FL IFITM2 or IFITM3. ( H ) Experiments were similar to the experiments in C except that anti-CD3 and anti-CD28 antibody-activated CD4 + T cells were treated with media, 1,000 U/mL IFN-β, or 1 μg/mL PHA for 2 d. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Providers Immunogens Protein recognized Anti-IFITM1 (goat) R&D Systems (catalog no. AF4827) IFITM1 Met1-His36 IFITM1 Anti-IFITM2 (mouse) Proteintech (catalog no. 66137-1-Ig) IFITM2 FL FL IFITM2 Anti-IFITM2/3 (goat) R&D Systems (catalog no. AF4834) IFITM3 His3-His57 FL IFITM2, IFITM3, Δ20 IFITM2 (weakly) Anti-IFITM2/3/Δ20 (rabbit) Cell Signaling Technology (catalog no. 13530) IFITM2 around pro41 FL IFITM2, IFITM3, Δ20 IFITM2 Anti-IFITM1/2/3 (rabbit) ProSci (catalog no. 5807) IFITM1 FL IFITM1, FL IFITM2, IFITM3, Δ20 IFITM2 Open in a separate window Properties of anti-IFITM antibodies used in our studies Like other IFITM proteins, Δ20 IFITM2 expression was up-regulated by IFNs in CD4 + T cells, moDCs, and macrophages, whereas phytohemagglutinin (PHA), which has been widely used for propagating HIV-1, depleted its expression ( and and ).

Techniques: Expressing, Plasmid Preparation, Western Blot, Labeling, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Δ20 IFITM2 differentially restricts X4 and R5 HIV-1

doi: 10.1073/pnas.1619640114

Figure Lengend Snippet: Properties of anti-IFITM antibodies used in our studies

Article Snippet: Note that the upper bands detected by the anti-IFITM2/3/Δ20 antibody could be FL IFITM2 or IFITM3. ( H ) Experiments were similar to the experiments in C except that anti-CD3 and anti-CD28 antibody-activated CD4 + T cells were treated with media, 1,000 U/mL IFN-β, or 1 μg/mL PHA for 2 d. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Providers Immunogens Protein recognized Anti-IFITM1 (goat) R&D Systems (catalog no. AF4827) IFITM1 Met1-His36 IFITM1 Anti-IFITM2 (mouse) Proteintech (catalog no. 66137-1-Ig) IFITM2 FL FL IFITM2 Anti-IFITM2/3 (goat) R&D Systems (catalog no. AF4834) IFITM3 His3-His57 FL IFITM2, IFITM3, Δ20 IFITM2 (weakly) Anti-IFITM2/3/Δ20 (rabbit) Cell Signaling Technology (catalog no. 13530) IFITM2 around pro41 FL IFITM2, IFITM3, Δ20 IFITM2 Anti-IFITM1/2/3 (rabbit) ProSci (catalog no. 5807) IFITM1 FL IFITM1, FL IFITM2, IFITM3, Δ20 IFITM2 Open in a separate window Properties of anti-IFITM antibodies used in our studies Like other IFITM proteins, Δ20 IFITM2 expression was up-regulated by IFNs in CD4 + T cells, moDCs, and macrophages, whereas phytohemagglutinin (PHA), which has been widely used for propagating HIV-1, depleted its expression ( and and ).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Δ20 IFITM2 differentially restricts X4 and R5 HIV-1

doi: 10.1073/pnas.1619640114

Figure Lengend Snippet: Characterization of the subcellular distribution of Δ20 IFITM2. (A) A549 cells transduced to express the indicated IFITM proteins were labeled with anti-LAMP2, anti-IFITM1, or anti-IFITM2/3 antibodies. Labeled cells were imaged by confocal microscopy. (B) Percentages of voxels of IFITM proteins localizing to the plasma membrane are shown. Errors bars denote 1 SD (n = 20). (C) Experiments were similar to the experiments in Fig. 1 F and G except that vector-transduced, IFITM1-expressing, or IFITM3-expressing Jurkat E6-1 R5 cells were labeled with anti-IFITM1 or anti-IFITM2/3 antibodies. (D) Percentages of voxels of IFITM proteins localizing to the plasma membrane are shown. Errors bars denote 1 SD (n = 20). (E) Experiments were similar to the experiments in C except that unactivated and anti-CD3 and anti-CD28 antibody-activated CD4+ T cells were labeled with an anti-IFITM2/3/Δ20 antibody.

Article Snippet: Note that the upper bands detected by the anti-IFITM2/3/Δ20 antibody could be FL IFITM2 or IFITM3. ( H ) Experiments were similar to the experiments in C except that anti-CD3 and anti-CD28 antibody-activated CD4 + T cells were treated with media, 1,000 U/mL IFN-β, or 1 μg/mL PHA for 2 d. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Providers Immunogens Protein recognized Anti-IFITM1 (goat) R&D Systems (catalog no. AF4827) IFITM1 Met1-His36 IFITM1 Anti-IFITM2 (mouse) Proteintech (catalog no. 66137-1-Ig) IFITM2 FL FL IFITM2 Anti-IFITM2/3 (goat) R&D Systems (catalog no. AF4834) IFITM3 His3-His57 FL IFITM2, IFITM3, Δ20 IFITM2 (weakly) Anti-IFITM2/3/Δ20 (rabbit) Cell Signaling Technology (catalog no. 13530) IFITM2 around pro41 FL IFITM2, IFITM3, Δ20 IFITM2 Anti-IFITM1/2/3 (rabbit) ProSci (catalog no. 5807) IFITM1 FL IFITM1, FL IFITM2, IFITM3, Δ20 IFITM2 Open in a separate window Properties of anti-IFITM antibodies used in our studies Like other IFITM proteins, Δ20 IFITM2 expression was up-regulated by IFNs in CD4 + T cells, moDCs, and macrophages, whereas phytohemagglutinin (PHA), which has been widely used for propagating HIV-1, depleted its expression ( and and ).

Techniques: Labeling, Confocal Microscopy, Clinical Proteomics, Membrane, Plasmid Preparation, Expressing