Journal: Virology

Article Title: S-palmitoylation of swine interferon-inducible transmembrane protein is essential for its anti-JEV activity

doi: 10.1016/j.virol.2020.06.004

Figure Lengend Snippet: Swine IFITM1 knockdown enhances JEV infection. (a) At 24h, 48h and 72h post-infection, the JEV genome copies number of culture supernatants of IFITM1 shRNA PK15 cells. (b) Quantitive RT-PCR analysis of sIFITM1 mRNA expression in control and IFITM1 shRNA PK15 cells. (c) Western blot analysis of whole cell extracts from control and IFITM1 shRNA PK15 cells infected with JEV. (d) At 24h, 48h and 72h post-infection, the JEV genome copies number of culture supernatants of IFITM1 shRNA ST cells. (e) Quantitive RT-PCR analysis of sIFITM1 mRNA in control and IFITM1 shRNA ST cells. (f) Western blot analysis of whole cell extracts from control and IFITM1 shRNA ST cells infected with JEV. Bar charts represent mean ± SEM of three experiments and each experiment included triplicate repeats. *p<0.05; **p <0.01; ***p <0.001, Student's t-test.

Article Snippet: Primary antibodies were as follows: rabbit anti-IFITM1 polyclonal antibody (Cusabio, China), monoclonal mouse anti-actin (Proteintech, Portland), mouse monoclonal anti-FLAG M2 antibody (Sigma-aldrich, USA).

Techniques: Knockdown, Infection, shRNA, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: Virology

Article Title: S-palmitoylation of swine interferon-inducible transmembrane protein is essential for its anti-JEV activity

doi: 10.1016/j.virol.2020.06.004

Figure Lengend Snippet: Biochemical analyses for S-palmitoylation on swine IFITM1. (a) The palmitoylation/depalmitoylation is a dynamic reversible reaction in which the palmitoyl thioester group is covalently connected to coenzyme A(CoA), is transferred to cysteine by palmitoyl acyl transferase (PAT). (b) The principle of acyl-PEGyl exchange gel shift. (c) Molecular structure of 2-bromopalmitic acid (2BP). (d) S-palmitoylated sIFITM1 was monitored through APEGS method mass-shift based detection. The cell lysates are incubated with TCEP, and free cysteine residues are protected with NEM. S-fatty acid groups of sIFITM1 are replaced by 5 KDa mPEG-Mal through two subsequent reactions, a reduction mediated by NH 2 OH results to cysteines exposure and then a ligation with mPEG-Mal. Proteins are subjected to SDS-PAGE and then measured by Western blot. The number 10 and 100 represented the μM concentration of 2-BP. The number of PEGylation exchange is indicated by asterisks (*). The lower two panels were SDS-PAGE of whole-cell lysate input fraction from cells used in upper APEGS analysis. Immunoblotting was performed with anti-FLAG and anti-actin. (e) The relative intensity of the palmitoylated and unpalmitolated sIFITM1. The relative intensity was a ratio of average intensity of estimates (n = 5) worked out according to the intensity of different mass-shift bands of sIFITM1 measured by Image Pro Plus software. The expression of actin and sIFITM1 in whole lysate were taken as important consideration in data processing.

Article Snippet: Primary antibodies were as follows: rabbit anti-IFITM1 polyclonal antibody (Cusabio, China), monoclonal mouse anti-actin (Proteintech, Portland), mouse monoclonal anti-FLAG M2 antibody (Sigma-aldrich, USA).

Techniques: Gel Shift, Incubation, Ligation, SDS Page, Western Blot, Concentration Assay, Software, Expressing

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