Journal: PLoS ONE

Article Title: Protective Roles of Interferon-Induced Protein with Tetratricopeptide Repeats 3 (IFIT3) in Dengue Virus Infection of Human Lung Epithelial Cells

doi: 10.1371/journal.pone.0079518

Figure Lengend Snippet: Human DCs (A) or A549 cells (B, C, and D) at 1 х 10 6 or 1 х 10 5 cells/mL were infected by mock or DV at various time points. Total cell lysates were collected and the expression of IFIT3 or β-actin was determined by western blotting (A and B) or immunocytochemical staining (C). Expression of mRNAs of ifit1 , ifit2 , ifit3 , and ifit5 genes was determined by quantitative RT-PCR (D). The data shown are from 3 independent experiments.

Article Snippet: After permeabilization with 1% Triton X-100 for 20 min, the cells were blocked with PBS containing 1% BSA and 0.1% Triton X-100 for 1 h, and then incubated sequentially for 2 h with anti-IFIT3 antibodies (rabbit monoclonal anti-human; GeneTex), followed by 1 h with a secondary antibody (Goat anti-rabbit IgG-FITC, at a 1:25 dilution; Abcam, Cambridge, ENG) at room temperature.

Techniques: Infection, Expressing, Western Blot, Staining, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Protective Roles of Interferon-Induced Protein with Tetratricopeptide Repeats 3 (IFIT3) in Dengue Virus Infection of Human Lung Epithelial Cells

doi: 10.1371/journal.pone.0079518

Figure Lengend Snippet: A549 cells were infected by mock or DV for 3, 6, and 24 h and protein levels of both phosphorylated and non-phosphorylated STAT1, STAT2, and STAT3 were analyzed by western blotting (A). Treatment with 1000 units IFN-α was used as a positive control. Expression of IFIT3 in DV-infected A549 cells with knockdown of either STAT2 (B), STAT1 (C) or STAT3 (D) was determined by western blotting (B, C, and D) or quantitative RT/PCR (B). Both shRNA and siRNA were used as the approaches for STAT1/STAT2 and STAT3, respectively, as described in Materials and Methods. Knockdown with shGFP or si-Ctl was used as a negative control. Data show representative results and analysis pooled from at least 3 independent experiments. The analysis was performed by ANOVA as described in Materials and Methods. **P < 0.01. Ctl stands for control.

Article Snippet: After permeabilization with 1% Triton X-100 for 20 min, the cells were blocked with PBS containing 1% BSA and 0.1% Triton X-100 for 1 h, and then incubated sequentially for 2 h with anti-IFIT3 antibodies (rabbit monoclonal anti-human; GeneTex), followed by 1 h with a secondary antibody (Goat anti-rabbit IgG-FITC, at a 1:25 dilution; Abcam, Cambridge, ENG) at room temperature.

Techniques: Infection, Western Blot, Positive Control, Expressing, Knockdown, Quantitative RT-PCR, shRNA, Negative Control, Control

Journal: PLoS ONE

Article Title: Protective Roles of Interferon-Induced Protein with Tetratricopeptide Repeats 3 (IFIT3) in Dengue Virus Infection of Human Lung Epithelial Cells

doi: 10.1371/journal.pone.0079518

Figure Lengend Snippet: A549 cells were transfected with different siRNAs (si-Ctl, siIFIT3-1, siIFIT3-2 or siIFIT3-3) for 24 h and then infected with mock or DV for 13 h. Expression of mRNA of ifit genes and IFIT3 protein was determined by quantitative RT/PCR and western blotting, respectively (A). Data show results of 3 independent experiments. A549 cells transfected with control siRNA (si-Ctl) or IFIT3 siRNA (siIFIT3-2) for 24 h were infected by mock or DV at M.O.I. = 0.5 or 5 for an additional 24 or 48 h. Cells were collected for measurement of expression of intracellular NS3 by flow cytometry (B). Supernatants were collected to determine virus titers by plaque assays (C). Data show results pooled from at least 3 independent experiments. The analysis was performed by ANOVA as described in Materials and Methods. **P < 0.01, ***P < 0.001. Ctl stands for control.

Article Snippet: After permeabilization with 1% Triton X-100 for 20 min, the cells were blocked with PBS containing 1% BSA and 0.1% Triton X-100 for 1 h, and then incubated sequentially for 2 h with anti-IFIT3 antibodies (rabbit monoclonal anti-human; GeneTex), followed by 1 h with a secondary antibody (Goat anti-rabbit IgG-FITC, at a 1:25 dilution; Abcam, Cambridge, ENG) at room temperature.

Techniques: Transfection, Infection, Expressing, Quantitative RT-PCR, Western Blot, Control, Flow Cytometry, Virus

Journal: PLoS ONE

Article Title: Protective Roles of Interferon-Induced Protein with Tetratricopeptide Repeats 3 (IFIT3) in Dengue Virus Infection of Human Lung Epithelial Cells

doi: 10.1371/journal.pone.0079518

Figure Lengend Snippet: A549 cells transfected with control siRNA (si-Ctl) or IFIT3 siRNA (siIFIT3) for 24 h were infected with mock or DV at M.O.I. = 0.5 or 5 for an additional 48 h. Cell viability was determined by MTT assays. Mock-infected cells treated with control siRNA transfection were taken as 100%, and OD values from individual conditions were normalized by the value of the control (% of control; A). DNA content was determined by sub-G1 analysis (left panel in B), and the synergistic effects were calculated (right panel in B). Determination and visualization of cell apoptosis were also performed by TUNEL assays using flow cytometry and immunofluorescent staining (C). Data show representative results and analysis pooled from at least 3 independent experiments. The analysis was performed by ANOVA as described in Materials and Methods. *P < 0.05, **P < 0.01, ***P < 0.001. Ctl stands for control.

Article Snippet: After permeabilization with 1% Triton X-100 for 20 min, the cells were blocked with PBS containing 1% BSA and 0.1% Triton X-100 for 1 h, and then incubated sequentially for 2 h with anti-IFIT3 antibodies (rabbit monoclonal anti-human; GeneTex), followed by 1 h with a secondary antibody (Goat anti-rabbit IgG-FITC, at a 1:25 dilution; Abcam, Cambridge, ENG) at room temperature.

Techniques: Transfection, Control, Infection, TUNEL Assay, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: Protective Roles of Interferon-Induced Protein with Tetratricopeptide Repeats 3 (IFIT3) in Dengue Virus Infection of Human Lung Epithelial Cells

doi: 10.1371/journal.pone.0079518

Figure Lengend Snippet: A549 cells transfected with control siRNA (si-Ctl) or IFIT3 siRNA (siIFIT3) for 24 h were infected by mock or DV at M.O.I. = 5 for another 24 h. The cleaved proteins, including caspase 8, caspase 9, caspase 3 and BAX were determined by western blotting (A). Caspase 3 activity (B) or Annexin V and 7-AAD (C) were determined by flow cytometry analysis at postinfection 48 h. The representative results and the analysis pooled from at least three independent experiments were shown. The analysis was performed by ANOVA as described in Materials and Methods. *P<0.05, **P<0.01, ***P<0.001. Ctl stands for control.

Article Snippet: After permeabilization with 1% Triton X-100 for 20 min, the cells were blocked with PBS containing 1% BSA and 0.1% Triton X-100 for 1 h, and then incubated sequentially for 2 h with anti-IFIT3 antibodies (rabbit monoclonal anti-human; GeneTex), followed by 1 h with a secondary antibody (Goat anti-rabbit IgG-FITC, at a 1:25 dilution; Abcam, Cambridge, ENG) at room temperature.

Techniques: Transfection, Control, Infection, Western Blot, Activity Assay, Flow Cytometry

Journal: PLoS ONE

Article Title: Protective Roles of Interferon-Induced Protein with Tetratricopeptide Repeats 3 (IFIT3) in Dengue Virus Infection of Human Lung Epithelial Cells

doi: 10.1371/journal.pone.0079518

Figure Lengend Snippet: A549 transfected with IFIT3-flag or empty vector (EV) for 24 h were infected by mock or DV at M.O.I. = 0.05 or 5 for 48 h. The expression of endogenous and exogenous IFIT3 was determined by western blotting as described in the Materials and Methods (A). The supernatants were collected for determining virus titers by plaque assays (B). After transfection, the cell were reseeded onto 96 well plate overnight and then infected by mock or DV at various M.O.I. = 6.25 to 200 for 48 h, the cell viability was determined by MTT assay (C). The representative results and the analysis pooled from at least three independent experiments are shown. The analysis was performed by student’s T test (B) or ANOVA (C) as described in Materials and Methods. *P<0.05.

Article Snippet: After permeabilization with 1% Triton X-100 for 20 min, the cells were blocked with PBS containing 1% BSA and 0.1% Triton X-100 for 1 h, and then incubated sequentially for 2 h with anti-IFIT3 antibodies (rabbit monoclonal anti-human; GeneTex), followed by 1 h with a secondary antibody (Goat anti-rabbit IgG-FITC, at a 1:25 dilution; Abcam, Cambridge, ENG) at room temperature.

Techniques: Transfection, Plasmid Preparation, Infection, Expressing, Western Blot, Virus, MTT Assay

Journal: PLoS ONE

Article Title: Protective Roles of Interferon-Induced Protein with Tetratricopeptide Repeats 3 (IFIT3) in Dengue Virus Infection of Human Lung Epithelial Cells

doi: 10.1371/journal.pone.0079518

Figure Lengend Snippet: Shortly after viral absorption, DV-infected A549 cells (1 x 10 5 /mL) were treated with 1000 units IFN-α and incubated for an additional 24 h. Alternatively, 1000 units IFN-α were added into the culture medium 12 h after virus infection and incubated for an additional 12 h. Expression of IFIT3 was determined by western blotting, and relative band intensities were quantified, right panel (A). IFN-α was added simultaneously with DV infection or 6 h or 12 h after DV infection. After incubation for additional 12 h, the supernatants were collected and virus titers were measured by plaque assays (B). Similar to (A), A549 cells infected by mock or DV for 12 h were treated with 100 units IFN-γ, and the cell cultures were then maintained for an additional 12 h. IFIT3 levels in cell lysates and virus titers in supernatants were determined (C). Data represent results from 3 independent experiments. The analysis was performed by ANOVA (A and B) or student’s T test (C) as described in Materials and Methods. *P < 0.05. n.s: no significance.

Article Snippet: After permeabilization with 1% Triton X-100 for 20 min, the cells were blocked with PBS containing 1% BSA and 0.1% Triton X-100 for 1 h, and then incubated sequentially for 2 h with anti-IFIT3 antibodies (rabbit monoclonal anti-human; GeneTex), followed by 1 h with a secondary antibody (Goat anti-rabbit IgG-FITC, at a 1:25 dilution; Abcam, Cambridge, ENG) at room temperature.

Techniques: Infection, Incubation, Virus, Expressing, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

doi: 10.1186/s13024-018-0263-7

Figure Lengend Snippet: Endogenous dsRNA response and type I interferon promote paraspeckle hyper-assembly in stable cell lines. a and b Depletion of TDP-43, Dicer, Drosha, ADAR1 but not Ago2 or FUS causes intracellular build-up of dsRNA. dsRNA was detected by immunocytochemistry using J2 antibody. Representative images of all conditions are shown. Scale bars, 50 and 10 μm for general plane and close-up panels respectively. c Levels of Alu-containing RNA as analysed by qRT-PCR using specific primers recognising Alu elements (n = 4). *p < 0.05 (Mann-Whitney U -test). d and e Markers of activated cellular reponse to dsRNA are upregulated in TDP-43 depleted cells. Levels of phosphorylated PKR and eIF2α were analysed by Western blot ( d , representative blots are shown) and expression of IFNB1 and an IFN-stimulated gene CXCL10 - by qRT-PCR ( e , n = 6). *p < 0.05 (Mann-Whitney U -test). f IFNbeta treatment stimulates NEAT1 expression and paraspeckle formation. NEAT1 levels were measured by qRT-PCR (n = 6). **p < 0.01 (Mann-Whitney U -test). Staining for an IFN-inducible protein IFIT3 was used as a positive control. Scale bar, 10 μm. g Simultaneous IFNbeta knockdown partially reverses the effect of TDP-43 depletion on paraspeckles. * and #p < 0.05, ***p < 0.001 (one-way ANOVA with Holm-Sidak correction for multiple comparisons). Scale bar, 10 μm. In all panels, cells were harvested for analysis 48 h post-transfection. Paraspeckles in panels f and g were visualised by NEAT1_2 RNA-FISH

Article Snippet: The following commercial primary antibodies were used: TDP-43 (rabbit polyclonal, 10782–2-AP, Proteintech and mouse monoclonal, MAB7778-SP, R&D Biosystems); FUS (rabbit polyclonal, Proteintech, 11570–1-AP); p54nrb/NONO (rabbit polyclonal C-terminal, Sigma); PSF/SFPQ (rabbit monoclonal, ab177149, Abcam); Tuj (β-Tubulin III, mouse monoclonal, Sigma); dsRNA (mouse monoclonal, J2, Kerafast); cleaved caspase 3 (rabbit polyclonal, 9661, Cell Signaling); NF-κB p65 (rabbit monoclonal, D14E12, Cell Signaling); IFIT3 (rabbit polyclonal, Bethyl); p-eIF2α (rabbit monoclonal, ab32157, Abcam); p-PKR (rabbit polyclonal, Thr451, ThermoFisher); PKR (mouse monoclonal, MAB1980-SP, R&D Systems); eIF2α (rabbit monoclonal, D7D3, Cell Signaling); β-actin (mouse monoclonal, A5441, Sigma).

Techniques: Stable Transfection, Immunocytochemistry, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Expressing, Staining, Positive Control, Knockdown, Transfection

Journal: Nature Communications

Article Title: A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

doi: 10.1038/s41467-024-52803-7

Figure Lengend Snippet: a WT and STING KO SCC25, H596, OE21, and Detroit 562 cells were subjected to RNA-seq analysis. Heatmap shows differentially expressed genes (absolute log 2 fold change of > 2 and an adjusted q value of < 0.05 from n = 4 independent replicates for each cell type) in the IFN and inflammatory pathways in any given cell line between the WT vs. STING KO variant. b–g RT-qPCR analysis of IFIT2, MX1 and IFIT3 in uninfected SCC25, H596, OE21 and Detroit 562 cells treated with increasing concentrations of SN-011 ( b – d ) and H-151 ( e – g ) for 48 h. Data show the mean from n = 2 independent biological replicates, error bars show SEM ( h , i ) WT and STING KO SCC25 and STING KO cells were pre-treated with 20 μM SN-011 ( h ) or H-151 ( i ) for 24 h were infected with WT SARS-CoV-2 at MOI: 1 i.u./cell for 72 h. Data show the copies of cell-associated SARS-CoV-2 N RNA in compound-treated cells normalized relative to mock-treated samples. Data show the mean from n = 3 independent biological replicates, error bars show SEM. Significance was assessed by multiple unpaired two-tailed t tests and p- values provided in the figure. Source data are provided as a Source Data file.

Article Snippet: After washing with PBST, samples were incubated in a goat anti-mouse secondary antibody conjugated to Alexa Fluor Plus 488 (Invitrogen, Cat# A-11029, 1:1000) at room temperature for 1 h. IFIT3, IFIT2, ISG15 and STING were detected by incubation with a primary rabbit polyclonal IFIT3 antibody (Novus Biologicals NBP2-32500, 1:500), rabbit polyclonal IFIT2 antibody (Novus Biologicals NBP2-15180SS, 1:500), rabbit polyclonal ISG15 antibody (Proteintech 15981-1-AP, 1:250) and rabbit polyclonal STING antibody (Proteintech 19851-1-AP, 1:200) respectively as described above followed by incubation with a goat anti-rabbit fluorescent secondary antibody (Invitrogen Goat anti-Rabbit Alexa Fluor Plus 568TM, Cat# A-11036, 1:1000 or Invitrogen Goat anti-Rabbit Alexa FluorTM Plus 647, Catalog # A32733TR, 1:1000).

Techniques: RNA Sequencing, Variant Assay, Quantitative RT-PCR, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

doi: 10.1038/s41467-024-52803-7

Figure Lengend Snippet: a Relative expression of ISGs including ISG15, IFIT1, IFIT2, IFIT3, and IRF7 in SCC25 cGAS KO, STING KO or NT control cells infected with SARS-CoV-2 at a MOI of 2 i.u./cell. Cells were collected at 72 hpi, and ISG expression was analyzed by RT-qPCR. Data show the fold induction of ISGs in infected over uninfected cells from n = 3 biological replicates, errors bars show the SEM. b , c SCC25 STING KO or NT control cells were infected with SARS-CoV-2 at an MOI of 2 i.u./cell. Immunofluorescence detection for IFIT3 expression (in red) and SARS-CoV-2 nucleocapsid (N) viral protein (in green), and cellular nuclei (DAPI, in blue) at 72 hpi from a representative experiment ( n = 2) are shown. Images were collected with an epifluorescence microscope, 4X objective ( b ), or Zeiss LSM 880 Airyscan confocal microscope equipped with a × 63/1.4 objective ( c ) as detailed in Methods. Scale bars = 250 μm ( b ) or 10 μm ( c ). d , e SCC25 STING KO cells were infected with SARS-CoV-2-mNG at MOI:1 i.u./cell, and cells were single-cell sorted at 72 hpi to isolate infected (mNG positive) and bystander cells as detailed in Methods. Expression of the indicated ISGs ( d ) and inflammatory genes ( e ) were analyzed by RT-qPCR in these sorted cell populations. Data show the mean from two independent replicates, error bars show the SEM. Source data are provided as a Source Data file.

Article Snippet: After washing with PBST, samples were incubated in a goat anti-mouse secondary antibody conjugated to Alexa Fluor Plus 488 (Invitrogen, Cat# A-11029, 1:1000) at room temperature for 1 h. IFIT3, IFIT2, ISG15 and STING were detected by incubation with a primary rabbit polyclonal IFIT3 antibody (Novus Biologicals NBP2-32500, 1:500), rabbit polyclonal IFIT2 antibody (Novus Biologicals NBP2-15180SS, 1:500), rabbit polyclonal ISG15 antibody (Proteintech 15981-1-AP, 1:250) and rabbit polyclonal STING antibody (Proteintech 19851-1-AP, 1:200) respectively as described above followed by incubation with a goat anti-rabbit fluorescent secondary antibody (Invitrogen Goat anti-Rabbit Alexa Fluor Plus 568TM, Cat# A-11036, 1:1000 or Invitrogen Goat anti-Rabbit Alexa FluorTM Plus 647, Catalog # A32733TR, 1:1000).

Techniques: Expressing, Control, Infection, Quantitative RT-PCR, Immunofluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

doi: 10.1038/s41419-021-03545-7

Figure Lengend Snippet: A Silencing of circUBAP2 was achieved in MHCC-97H and Huh-7 cells by transfecting small interfering RNA targeting circUBAP2 (si-circUBAP2-1 or si-circUBAP2-2). Si-NC was transfected as a negative control. The transfection efficiency was confirmed by real-time PCR and si-circUBAP2-1 was chosen for further experiments for its better transfection efficiency. Then, MHCC-97H and Huh-7 cells were transfected with si-circUBAP2 and examined for B Cell migration by Transwell assay; C Cell migration by wound healing assay; D The protein levels of Vimentin and Twist was examined by Immunoblotting; E The concentrations of IL-1β and IL-17 in the culture medium by ELISA. F , G The Volcano plot and hierarchical clustering heatmap showing differentially expressed mRNAs in CXCL11-treated MHCC-97H based on RNA sequencing. Upregulated genes were applied for H Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling enrichment analysis and I Gene Ontology (GO) of biological process enrichment analysis. J The expression of IFIT1/IFIT3 in CXCL11-treated and untreated HCC cells based on RNA sequencing data. * P < 0.05, ** P < 0.01, compared with the control group; # P < 0.05, ## P < 0.01, compared with the si-NC group; $ P < 0.05, $$ P < 0.01, compared with the CXCL11 + si-circUBAP2 group.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio).

Techniques: Small Interfering RNA, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Migration, Transwell Assay, Wound Healing Assay, Western Blot, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Expressing, Control

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

doi: 10.1038/s41419-021-03545-7

Figure Lengend Snippet: A The mRNA expression of IFIT1 and IFIT3 was examined in 12 cases of nonmetastatic HCC tissues, metastatic HCC tissues, and para-carcinoma tissues by real-time PCR. B The protein contents and distribution of IFIT1 and IFIT3 were examined in nonmetastatic HCC tissues, metastatic HCC tissues, and para-carcinoma tissues by IHC staining. C MHCC-97H and Huh-7 cells were transfected with si-circUBAP2 and examined for the mRNA expression of IFIT1 and IFIT3 by real-time PCR. D IFIT1 or IFIT3 silencing was achieved in MHCC-97H and Huh-7 cells by transfecting small interfering RNA targeting IFIT1 or IFIT3 (si-IFIT1-1 or si-IFIT1-2; si-IFIT3-1 or si-IFIT3-2). Si-NC was transfected as a negative control. The transfection efficiency was confirmed by real-time PCR and si-IFIT3-1 and si-IFIT1-2 were chosen for further experiments due to better transfection efficiency. Next, MHCC-97H and Huh-7 cells were transfected with si-NC, si-IFIT1, or si-IFIT3, and examined for E Cell migration by Transwell assay; F Cell migration by wound healing assay; G Cellular protein content and distribution of Vimentin was examined by IF staining; H The protein levels of Vimentin and Twist was examined by Immunoblotting; I The concentrations of IL-1β and IL-17 in the culture medium by ELISA. * P < 0.05, ** P < 0.01, compared with the si-NC group.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Transfection, Small Interfering RNA, Negative Control, Migration, Transwell Assay, Wound Healing Assay, Staining, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

doi: 10.1038/s41419-021-03545-7

Figure Lengend Snippet: A ENCORI was used to predict miRNAs that circUBAP2 might target, and TargetScan was used to predict miRNAs that might target IFIT1/IFIT3. These two sets of miRNAs intersected at hsa-miR-4756-5p, hsa-miR-1321, hsa-miR-552-3p, hsa-miR-3611, and hsa-miR-24-3p. The expression of miR-4756-5p, miR-1321, miR-552-3p, miR-3611, and miR-24-3p was examined in MHCC-97H and Huh-7 cells with or without CXCL11 treatment. miR-4756 was chosen for further experiments due to its downregulation in both HCC cell lines in response to CXCL11 stimulation. B MHCC-97H and Huh-7 were transfected with si-circUBAP2 and examined for the expression of miR-4756 by real-time PCR. C The expression of miR-4756 was examined in 12 cases of nonmetastatic HCC tissues, metastatic HCC tissues, and para-carcinoma tissues by real-time PCR. D miR-4756 overexpression or inhibition was achieved in MHCC-97H and Huh-7 cells by transfecting miR-4756 mimics or miR-4756 inhibitor; the transfection efficiency was confirmed by real-time PCR. E MHCC-97H and Huh-7 cells were transfected with miR-4756 mimics or miR-4756 inhibitor and examined for the protein levels of IFIT1 and IFIT3 by Immunoblotting. F – H Wild- and mutant-type circUBAP2, IFIT1 3′-UTR, or IFIT3 3′-UTR luciferase reporter plasmids were constructed as described and named as wt-circUBAP2/mut-circUBAP2, wt-IFIT1/mut-IFIT1, and wt-IFIT3/mut-IFIT3. These plasmids were co-transfected in 293T cells with miR-4756 mimics or miR-4756 inhibitor and the luciferase activity was determined. * P < 0.05, ** P < 0.01, compared to si-NC + inhibitor NC group. ## P < 0.01, compared to si-cirUBAP2+miR-4756 inhibitor group.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Over Expression, Inhibition, Western Blot, Mutagenesis, Luciferase, Construct, Activity Assay

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

doi: 10.1038/s41419-021-03545-7

Figure Lengend Snippet: MHCC-97H and Huh-7 cells were co-transfected with si-circUBAP2 and miR-4756 inhibitor and examined for A The protein levels of IFIT1 and IFIT3 by Immunoblotting; B The concentrations of IL-1β, and IL-17 in the culture medium by ELISA; C Cell migration by Transwell assay; D Cell migration by wound healing assay; E The cellular protein content and distribution of Vimentin was examined by IF staining; F The protein levels of Vimentin and Twist was examined by Immunoblotting. * P < 0.05, ** P < 0.01, compared with the si-NC + inhibitor NC group; ## P < 0.01, compared with the si-circUBAP2 + miR-4756 inhibitor group.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio).

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay, Wound Healing Assay, Staining

Journal: Scientific Reports

Article Title: Gene Expression Profiling Identifies Interferon Signalling Molecules and IGFBP3 in Human Degenerative Annulus Fibrosus

doi: 10.1038/srep15662

Figure Lengend Snippet: Microarray gene expression comparison of human annulus fibrosus cells.

Article Snippet: For immunohistochemistry deparaffinised sections were first treated with 0.3% hydrogen peroxide in methanol for 30 min. Antigen retrieval was performed by heating the sections (95 °C) in citrate buffer (pH 6.0) for 15 min. Then sections were blocked with 5% normal goat serum for 1 h and treated with anti-IGFBP3 antibody (Acris Antibodies, cat. nr. AP14347PU-N; 6.25 μg/mL) or anti-IFIT3 antibody (Aviva Systems, cat. nr. ARP46034_P050; 5 μg/mL) for 30 min at room temperature.

Techniques: Microarray, Gene Expression, Comparison, Binding Assay

Journal: Scientific Reports

Article Title: Gene Expression Profiling Identifies Interferon Signalling Molecules and IGFBP3 in Human Degenerative Annulus Fibrosus

doi: 10.1038/srep15662

Figure Lengend Snippet: Real-time RT-PCR data confirm IGFBP3, IFIT3, TFPI and IFIT2 were significantly increased in degenerative annulus fibrosus cells, while PMAIP1, GDF15, MGST1 and IFIT1 were up-regulated with no statistical significance. The data represent fold changes ± standard error. For statistical analysis, a two-tailed t-test was carried out on log 2 transformed relative expression data and multiple testing correction was performed according to the method of Benjamini and Hochberg, with 10% false discovery rate considered significant. N = 10 for degenerative samples; N = 8 for non-degenerative samples; *p < 0.05; #p < 0.1 degenerative versus non-degenerative.

Article Snippet: For immunohistochemistry deparaffinised sections were first treated with 0.3% hydrogen peroxide in methanol for 30 min. Antigen retrieval was performed by heating the sections (95 °C) in citrate buffer (pH 6.0) for 15 min. Then sections were blocked with 5% normal goat serum for 1 h and treated with anti-IGFBP3 antibody (Acris Antibodies, cat. nr. AP14347PU-N; 6.25 μg/mL) or anti-IFIT3 antibody (Aviva Systems, cat. nr. ARP46034_P050; 5 μg/mL) for 30 min at room temperature.

Techniques: Quantitative RT-PCR, Two Tailed Test, Transformation Assay, Expressing

Journal: Scientific Reports

Article Title: Gene Expression Profiling Identifies Interferon Signalling Molecules and IGFBP3 in Human Degenerative Annulus Fibrosus

doi: 10.1038/srep15662

Figure Lengend Snippet: Immunohistochemical analysis of IGFBP3 ( a–c ) and IFIT3 ( d–f ) in sections of human degenerative ( a, b, d, e ) and non-degenerative ( c, f ) annulus fibrosus. Intense immunolabelling for IGFBP3 ( a ) and IFIT3 ( d ) was observed in degenerated regions of the annulus fibrosus of a lumbar disc from a 47 year old female with disc degeneration Grade III. Negative control sections for IGFBP3 ( b ) and IFIT3 ( e ) did not show any staining. IGFBP3 positive cells were absent ( c ), while IFIT3 positive cells were observed in the inner annulus fibrosus of a normal disc ( f ) (degeneration Grade I) from a 13 year old male. Scale bar: 50 μm.

Article Snippet: For immunohistochemistry deparaffinised sections were first treated with 0.3% hydrogen peroxide in methanol for 30 min. Antigen retrieval was performed by heating the sections (95 °C) in citrate buffer (pH 6.0) for 15 min. Then sections were blocked with 5% normal goat serum for 1 h and treated with anti-IGFBP3 antibody (Acris Antibodies, cat. nr. AP14347PU-N; 6.25 μg/mL) or anti-IFIT3 antibody (Aviva Systems, cat. nr. ARP46034_P050; 5 μg/mL) for 30 min at room temperature.

Techniques: Immunohistochemical staining, Negative Control, Staining

Journal: Scientific Reports

Article Title: Gene Expression Profiling Identifies Interferon Signalling Molecules and IGFBP3 in Human Degenerative Annulus Fibrosus

doi: 10.1038/srep15662

Figure Lengend Snippet: The schematic generated is representative of the figure produced by IPA ingenuity pathway analysis system that shows the most dysregulated canonical pathway, the IFN signalling pathway, identified by microarray data of human degenerative versus non-degenerative annulus fibrosus. Several genes found dysregulated in degenerative annulus fibrosus are induced or up-regulated through IFNA signalling pathway, such as IFIT1, IFIT2, IFIT3 , as well as IGFBP3 and are indicated in pink.

Article Snippet: For immunohistochemistry deparaffinised sections were first treated with 0.3% hydrogen peroxide in methanol for 30 min. Antigen retrieval was performed by heating the sections (95 °C) in citrate buffer (pH 6.0) for 15 min. Then sections were blocked with 5% normal goat serum for 1 h and treated with anti-IGFBP3 antibody (Acris Antibodies, cat. nr. AP14347PU-N; 6.25 μg/mL) or anti-IFIT3 antibody (Aviva Systems, cat. nr. ARP46034_P050; 5 μg/mL) for 30 min at room temperature.

Techniques: Generated, Produced, Microarray