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: The Journal of investigative dermatology

Article Title: Delineating the Healthy Human Skin UV Response and Early Induction of Interferon Pathway in Cutaneous Lupus Erythematosus.

doi: 10.1016/j.jid.2019.02.035

Figure Lengend Snippet: Figure 2. Altered UV photoprovocation-response genes in DLE and SCLE. (a) The upregulated genes (yellow box) in the known SLE pathway [KEGG: hsa05322]. (b) Ten most enriched gene sets in the LINCS L1000 ligand perturbation dataset and the altered genes corresponding to the enriched gene sets; each gene set consists of differentially regulated genes induced by a ligand in a cell line. (c) Upregulation of IFIT1, IFIT2, and IFIT3 mRNA expression in photoprovocated SCLE (in all but two) and DLE skin lesions by qRT-PCR validation. (d) IFIT protein expression in skin samples by immunohistochemistry. IFIT proteins are more strongly expressed in SCLE than in healthy controls after UV provocation. Note the strong expression (dark brown) especially in basal keratinocytes and in areas with apoptotic keratinocytes (i.e., cells with pyknotic nuclei and eosinophilic cytoplasm). Scale bar ¼ 100 mm. DLE, discoid lupus erythematosus; qRT-PCR, quantitative real-time reverse transcriptaseePCR; SCLE, subacute cutaneous lupus erythematosus; SLE, systemic lupus erythematosus.

Article Snippet: Primary polyclonal rabbit antibodies to IFIT1, IFIT2, and IFIT3 (Sigma/Atlas antibodies, St. Louis, MO) were diluted in 1% BSA and applied on the sections overnight at 4 C. The bound antibodies were visualized using Vector Universal ImmPress Kit and Vector NovaRed (Vector Laboratories, Burlingame, CA), and counterstained with hematoxylineeosin.

Techniques: Expressing, Quantitative RT-PCR, Biomarker Discovery, Immunohistochemistry

Journal: Nature neuroscience

Article Title: CRISPRi screens in human iPSC-derived astrocytes elucidate regulators of distinct inflammatory reactive states

doi: 10.1038/s41593-022-01180-9

Figure Lengend Snippet: a, Transcript levels of IFIT3 overlaid onto the UMAP embedding from Fig. 3a. b, Representative immunofluorescence images of C3 and IFIT3 staining (scale bar: 60 μm). c, Percent IFIT3−/C3+, IFIT3+/C3−, or IFIT3+/C3+ cells measured by immunofluorescence in iAstrocytes derived from multiple hiPSC lines (WTC11, TCW-1E44, 162D) treated with vehicle control vs. all possible combinations of IL-1α, TNF, and C1q, in the absence (n = 3 wells per condition) or presence of additional IL-6/IL6R chimera (25 ng/mL) or IFN-β (5 ng/mL) added concurrently (n = 4 wells per condition). D, Concentration of IFN-β, IL-6, CXCL10, or GM-CSF in conditioned media from iAstrocytes derived from multiple hiPSC lines (WTC11, 162D) treated with vehicle control vs. all possible combinations of IL-1α, TNF, and C1q (n = 4 wells). For panels c a, P values were calculated using beta regression (two-sided Wald test; see Methods). For panel d, P values were calculated using linear regression (two-sided Wald test; see Methods). P values were adjusted for multiple testing (Padj; Holm’s method) per family of tests (all comparisons within a plot).

Article Snippet: For immunostaining of C3 and IFIT3, iAstrocytes derived from WTC11, TCW-1E44, and 162D hiPSCs washed, fixed, and blocked as above and then incubated with primary antibodies against C3 (1:250, mouse monoclonal; BioLegend cat. no. 846302), IFIT3 (1:500, rabbit polyclonal; ThermoFisher Scientific cat. no. PA5-22230), or Na + /K + ATPase (1:500, chicken polyclonal; VWR cat. no. 89162–626) overnight at 4 °C in blocking buffer.

Techniques: Immunofluorescence, Staining, Derivative Assay, Concentration Assay

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: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: Co-precipitation assays between IFIT1 and (A) IFIT2, (B) IFIT5, and (C–D) IFIT3. MBP-tagged IFIT proteins were incubated with amylose resin (lane 1) and unbound MBP-tagged protein was washed (lanes 2–5). Prebound MBP-tagged IFIT (lane 6) was incubated with untagged IFIT proteins (lane 7) prior to washes (lane 8–11) and final beads (lane 12). Bound protein was eluted with maltose-containing buffer (lane 13). Final beads and eluted samples (boxed) were assessed for binding. Data shown are representative of at least two independent experiments. (E) Structure and domain organization of IFIT1 bound to 5′ cap 0 RNA (left), including location of TPR regions. Cartoon representation of IFIT1 colored by subdomain (right). (F) Cartoon representation of IFIT1-FIT3-ap 0 RNA heterotrimeric complex structure: asymmetric unit shows IFIT1 molecule A (green), IFIT1 molecule B (cyan) bound to IFIT3CTD molecule C (magenta), IFIT3CTD molecule D (yellow), and 5′ cap 0 RNA (orange). See also Figure S1 and S2.

Article Snippet: Immunoblots were performed using a rabbit polyclonal anti-IFIT1 (Thermo Fisher), rabbit anti-GAPDH (Cell Signaling Technology), rabbit polyclonal anti-IFIT3 ( Fensterl et al., 2008 ), or mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), followed by IRDye 680RD goat anti-rabbit or IRDye 800CW goat anti-mouse (LiCor).

Techniques: Incubation, Binding Assay

Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: (A) The molecular interface between IFIT1 and IFIT3 is defined by HDX-MS. Differences in deuterium uptake induced by IFIT3 binding are displayed as a color gradient (see legend) and highlighted in the cartoon representation of IFIT1. (B) Comparison of deuterium uptake kinetic curves of IFIT1 (black) and IFIT1-IFIT3 (green). Representative ITC raw data and binding isotherm for (C) IFIT1 and IFIT3. Measured values are KD = 1.5 ± 2.0 nM, ΔH = − 1.3 ± 0.02 × 104 cal/mol, ΔS = − 2.19 cal/mol/deg, and n (no. of sites) = 0.93 ± 0.01. (D) IFIT1 and IFIT3ΔCTD. KD = not determined. (E) IFIT1 and IFIT3CTD. Measured values are KD = 1.4 ± 3.1 nM, ΔH = − 1.7 ± 0.06 × 104 cal/mol, ΔS = − 2.08 cal/mol/deg., and n (number of sites) = 0.97 ± 0.03. (F) IFIT1 and IFIT3CTD K426A-E439A-L445A-S451A-I453A-F457A mutant (IFIT3 CTD mut). KD = not determined. All experiments were performed at least four independent times. See also Figure S3 and S4.

Article Snippet: Immunoblots were performed using a rabbit polyclonal anti-IFIT1 (Thermo Fisher), rabbit anti-GAPDH (Cell Signaling Technology), rabbit polyclonal anti-IFIT3 ( Fensterl et al., 2008 ), or mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), followed by IRDye 680RD goat anti-rabbit or IRDye 800CW goat anti-mouse (LiCor).

Techniques: Binding Assay, Mutagenesis

Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: (A) Comparison of 5′-ppp RNA-bound IFIT5 (green) (PDB 4HOR) and IFIT1 bound to cap 0 RNA and IFIT3CTD (cyan; (primary PDB validation is provided), current structure). (B) Structural changes on IFIT1 upon IFIT3 binding as defined by HDX-MS. Differences in deuterium uptake induced by IFIT3 binding to IFIT1-cap 0 RNA complex are displayed as a color gradient (see legend) and highlighted in the cartoon representation of IFIT1. Data shown are representative of two independent experiments. Multiple peptides were identified by MS/MS, and only the ten highest abundant peptides were used in the analysis. (C) Quantitative filter binding assay for IFIT1 (solid) and IFIT1-IFIT3CTD (dotted) and cap 0 RNA (black), cap1 RNA (red), and 5′-ppp RNA (blue). The results are the average of at least three independent experiments. Error bars represent standard error of the mean (SEM). See also Figure S5.

Article Snippet: Immunoblots were performed using a rabbit polyclonal anti-IFIT1 (Thermo Fisher), rabbit anti-GAPDH (Cell Signaling Technology), rabbit polyclonal anti-IFIT3 ( Fensterl et al., 2008 ), or mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), followed by IRDye 680RD goat anti-rabbit or IRDye 800CW goat anti-mouse (LiCor).

Techniques: Binding Assay, Tandem Mass Spectroscopy, Filter-binding Assay

Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: IFIT3 gene edited (IFIT3mut/mut) 293T cells were trans-complemented with IFIT3 (IFIT3mut/mut + IFIT3), IFIT3 lacking the C-terminal tail (IFIT3mut/mut + IFIT3CTD), or firefly luciferase (IFIT3mut/mut + fluc). (A) 293T-IFIT3mut/mut + IFIT3 and 293T-IFIT3mut/mut + fluc cells were infected at an MOI of 5 with WNV WT or isogenic WNV NS5-E218A lacking 2′-O methylation of the cap structure of genomic RNA. Infection was measured 24 hpi by flow cytometry by staining for intracellular WNV E protein. The fraction of infected 293T-IFIT3mut/mut + IFIT3 cells relative to infected 293T-IFIT3mut/mut + fluc are shown for both viruses. Data are the mean of three independent experiments, and error bars represent SEM. (B–C) 293T-IFIT3mut/mut + IFIT3 and 293T-IFIT3mut/mut + fluc cells were infected with WNV WT or WNV NS5-E218A at an MOI of 0.001. Supernatant was collected at the indicated times after infection and analyzed by focus-forming assay. Data are the mean titers from six independent experiments, and errors bars represent SEM. (D) IFIT3mut/mut + IFIT3, IFIT3mut/mut + hIFIT3ΔCTD, IFIT3mut/mut + mIfit3CTD, and IFIT3mut/mut + fluc cells were stimulated with IFN-β and assessed for IFIT1 expression by Immunoblotting. One representative experiment of three is shown. (E–F) 293T cells expressing human IFIT1-flag under an inducible promoter (293T-IFIT1-doxy) were trans-complemented with IFIT3 or fluc and analyzed for IFIT1 expression (anti-flag tag) by Immunoblotting. (E) One of two representative Immunoblots is shown. (F) IFIT1 amounts were normalized to GAPDH in IFIT3 or fluc-transduced cells treated with doxycycline. Error bars represent SEM from two independent experiments. (G–H) 293T-IFIT1-doxy cells trans-complemented with IFIT3 or fluc were stimulated with doxycycline for 16 h at indicated concentrations, subsequently treated with 50 μM puromycin to arrest translation, and analyzed for IFIT1 (anti-flag tag) at the indicated time post-puromycin treatment. (G) One of three representative Immunoblots is shown. (H) IFIT1 protein amounts were quantified and normalized to the 0 h post-puromycin treatment. Error bars represent SEM from three independent experiments. The statistical significance shown is for the comparison of IFIT3 + 100 ng/ml doxy vs fluc + 100 ng/ml doxy; comparisons between other doses yielded similar results. (I-K) CRISPR-Cas9 gene editing was used to abolish IFIT1 expression from 293T-IFIT3mut/mut + IFIT3 and 293T-IFIT3mut/mut + fluc cells using two different IFIT1-targeting sgRNAs or as a control, scrambled sgRNAs. (I) IFIT1 expression following IFN-β stimulation was determined by Immunoblot. (J–K) 293T-IFIT3mut/mut + IFIT3 and 293T-IFIT3mut/mut + fluc cells that received IFIT1 or scrambled sgRNAs were infected with WNV WT or WNV NS5-E218A at an MOI of 5 (J) or WT or NS5-E218A ZIKV (Cambodia, 2010; strain FSS13025) at an MOI or 1 (K) and scored for infectivity at 24 (J) or 30 (K) hpi by flow cytometry. Infectivity was normalized to the fraction of infected 293T-IFIT3mut/mut + fluc cells that received scrambled sgRNAs. The mean relative infectivity from three (K) and five (J) independent experiments is shown, and error bars represent the SEM. In this Figure, statistical significance was determined using an ANOVA followed by Tukey’s (A, B, and C) or Sidak’s (H, and J–K) multiple comparisons tests (ns, not significant; *, P > 0.05; ***, P <0.001; ****, P < 0.0001). See also Figure S6.

Article Snippet: Immunoblots were performed using a rabbit polyclonal anti-IFIT1 (Thermo Fisher), rabbit anti-GAPDH (Cell Signaling Technology), rabbit polyclonal anti-IFIT3 ( Fensterl et al., 2008 ), or mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), followed by IRDye 680RD goat anti-rabbit or IRDye 800CW goat anti-mouse (LiCor).

Techniques: Luciferase, Infection, Methylation, Flow Cytometry, Staining, Focus Forming Assay, Expressing, Western Blot, FLAG-tag, CRISPR

Journal: Immunity

Article Title: Binding of IFIT1 by IFIT3 modulates protein stability and RNA binding specificity

doi: 10.1016/j.immuni.2018.01.014

Figure Lengend Snippet: (A, B and E) 293T-IFIT1-doxy cells were transfected with plasmids encoding fluc, IFIT3, or the indicated IFIT3 mutants, and treated with doxycycline prior to infection with (A) WNV WT or (B and E) WNV NS5 E218A. Infection and transfection data were analyzed by flow cytometry 24 hpi (A and B, left panels; and also see Fig S6E) to determine the percentage of transfected cells that were positive for intracellular WNV E protein. Data are representative of three independent experiments, and error bars represent SEM. (A and B, right panels, and E) Data are normalized to infectivity of doxycycline-untreated cells for each independent transfection (IFIT3, IFIT3 mutants, or fluc) experiment. Errors bars represent the SEM, and data was pooled from three to six independent experiments for statistical analysis (below). (C–D) 293T-IFIT1-doxy cells were trans-complemented with IFIT3 or fluc and infected with (C) VEEV-TC83 (MOI of 1, 16 h) or (D) VSV (MOI of 1, 6 h). Left panels show the mean percentage of infected cells from one representative experiment, and error bars indicate the SEM from triplicate replicates. Right panels show data normalized to infectivity of doxycycline-untreated cells. Errors bars represent the SEM, and data was pooled from three independent experiments. In this Figure, statistical significance was determined using a t-test (A) or an ANOVA followed by Sidak’s (B, C, and D) or Dunnett’s (E) multiple comparisons tests (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). See also Figure S6.

Article Snippet: Immunoblots were performed using a rabbit polyclonal anti-IFIT1 (Thermo Fisher), rabbit anti-GAPDH (Cell Signaling Technology), rabbit polyclonal anti-IFIT3 ( Fensterl et al., 2008 ), or mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), followed by IRDye 680RD goat anti-rabbit or IRDye 800CW goat anti-mouse (LiCor).

Techniques: Transfection, Infection, Flow Cytometry

Journal: iScience

Article Title: Glucocorticoid receptor-mediated Nr1d1 chromatin circadian misalignment in stress-induced irritable bowel syndrome

doi: 10.1016/j.isci.2023.107137

Figure Lengend Snippet:

Article Snippet: IFIT3 Rabbit pAb , ABclonal , A3924.

Techniques: Recombinant, Reporter Assay, Knock-Out, Software

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