Journal: PLoS Pathogens

Article Title: Ebola virus glycoprotein directly triggers T lymphocyte death despite of the lack of infection

doi: 10.1371/journal.ppat.1006397

Figure Lengend Snippet: A , B . Western blotting analysis of immunoprecipitation of TLR4 or TLR-4 FLAG with EBOV GP (A) or EBOV VP40 (B). Representative data from one of two independent experiments. C. Confocal microscopy of HPIV3/ΔF-HN/EboGP bound to primary CD4 + T lymphocytes, Jurkat cells and 293T-TLR4 cells. Insets show the formation of plasma membrane associated GP-positive puncti. D. Flow cytometry analysis demonstrating the binding of EBOV and HPIV3/ΔF-HN/EboGP to SupT1 T cells. E. Flow cytometry analysis of TLR4 expression by isolated CD4 + T cells. T cells were activated with CD3/CD28 beads and then cultured with EBOV. Results are representative of 3 donors. F . Inhibition of EBOV binding to SupT1 cells by anti-TLR4 serum: % of no serum control. Mean values ±SE based on triplicate samples of one of two independent experiments, *** P<0.001 (Student T-test).

Article Snippet: GP-TLR4 binding was determined by co-transfecting 293T cells with mammalian codon-optimized plasmids encoding EBOV GP or VP40, as well as the plasmids expressing TLR4 (Addgene, #20863) or TLR4 FLAG-tagged protein (TLR4-FLAG) (Addgene, #42646) using TransIT-LT1 reagent (Mirus) for 48 h at 37°C.

Techniques: Western Blot, Immunoprecipitation, Confocal Microscopy, Flow Cytometry, Binding Assay, Expressing, Isolation, Cell Culture, Inhibition

Journal: PLoS Pathogens

Article Title: Ebola virus glycoprotein directly triggers T lymphocyte death despite of the lack of infection

doi: 10.1371/journal.ppat.1006397

Figure Lengend Snippet: A, B. Western blot analysis of proteins involved in TLR4 signaling pathway TRAM1, p-TRAM1, MyD88, IRAK4, p-IRAK4, Pyk2, p-Pyk2, p38, p-p38 in SupT1 cells and monocytes (A) or THP-1, THP-1 MyD88-/- and SupT1 cells (B) following stimulations with LPS, poly I:C, HPIV3/EboGP, HPIV3/ΔF-HN/EboGP, HPIV3 or EBOV (A) or empty beads or EBOV GP beads (B) in the presence or absence of the TLR4 inhibitor CLI-095. C , D , E . Western blot analysis of p65 phosphorylation in SupT1 cells (C), monocytes (D) or THP-1, THP-1 MyD88-/- and SupT1 cells (E) following stimulation with CD3/CD28 beads, LPS (+, 100 ng/ml, ++; 500 ng/ml), VLP (+, 100 μl; ++, 250 μl) and HPIV3/ΔF-HN/EboGP (+, MOI 0.1 PFU/cell; ++, MOI 1 PFU/cell), empty beads or EBOV GP beads, as indicated, with or without CLI-095. Western blots in panels A-E are representative of two independent experiments. F . Percentages of dead (Live/Dead + ) cells, cell positive for caspase 8 and 9 and proliferated CD4 + T lymphocytes following a 4 day-long incubation with EBOV or LPS with or without CLI-095. Mean values ±SE based on triplicates from one of two independent experiments with P values * P <0.05, ** P <0.01, n.s., non-significant (Student T-test).

Article Snippet: GP-TLR4 binding was determined by co-transfecting 293T cells with mammalian codon-optimized plasmids encoding EBOV GP or VP40, as well as the plasmids expressing TLR4 (Addgene, #20863) or TLR4 FLAG-tagged protein (TLR4-FLAG) (Addgene, #42646) using TransIT-LT1 reagent (Mirus) for 48 h at 37°C.

Techniques: Western Blot, Incubation

Journal: The Journal of investigative dermatology

Article Title: Integrin α4β1 and TLR4 Cooperate to Induce Fibrotic Gene Expression in Response to Fibronectin’s EDA Domain

doi: 10.1016/j.jid.2017.08.005

Figure Lengend Snippet: Fibroblasts were pre-treated with blocking antibodies (10 µg/ml) to the α4β1 integrin (MAB16983) or TLR4 for 30 minutes. Cells were then plated on mixtures of pFn (10 μg/ml) and EDA (40 μg/ml) or the control module (C) III-10n (40 μg/ml) in 0.1% BSA-DMEM for 2 hours. RNA was extracted and VCAM1 (a) and TNF (b) mRNA levels were assessed by real time RT-PCR. Fibroblasts were transiently transfected with 10 nM α4 siRNA or 10 nM non-targeting (NT) siRNA for 4 days. (c) Transfected cells were assessed for α4 knockdown by western blot both before and after replating onto Fn substrates. Cells were pre-treated with 1 μM TLR4 inhibitor (TAK 242), 1 μM NF-kB inhibitor (Bay 11–0872) or DMSO prior to being seeded onto mixtures of pFn (10 μg/ml) and EDA or III-10n (40 μg/ml) in 0.1% BSA-DMEM for 2 hours. RNA was extracted and (d) VCAM1 and TNF or (e) IL-10 and IL13 mRNA levels were assessed by real time RT-PCR. Fold change values are expressed relative to non-targeting (NT) siRNA treated cells plated onto pFn and III-10n. Error bars indicate standard error of quadruplicate samples for one of three representative experiments. Statistical analysis was performed using a two-way ANOVA with Tukey post-hoc test. In VCAM and IL-10 experiments, *P<0.05, **P<0.01 for α4 knockdown cells, TAK and Bay treated cells plated to EDA. In TNF and IL-13 experiments, #P<0.05, ##P<0.01 for α4 knockdown cells and TAK treated cells compared with NT siRNA treated cells plated onto pFn and III-10n.

Article Snippet: Blocking antibody to human TLR4 was obtained from R&D Systems (Minneapolis, MN).

Techniques: Blocking Assay, Quantitative RT-PCR, Transfection, Western Blot

Journal: The Journal of investigative dermatology

Article Title: Integrin α4β1 and TLR4 Cooperate to Induce Fibrotic Gene Expression in Response to Fibronectin’s EDA Domain

doi: 10.1016/j.jid.2017.08.005

Figure Lengend Snippet: (a) Prior to seeding cells onto substrates, cells were incubated with blocking antibody (10 μg/ml) to the α4β1 integrin (α4) or TLR4. Mouse IgG (IgG) was used as a control. Real time RT-PCR analysis of EDA+Fn, Total Fn, as well as β-Actin mRNA was assessed. (b) Fibroblasts were transiently transfected with 10 nM α4 siRNA or 10 nM non-targeting (NT) siRNA for 4 days. Transfected cells were pre-treated with 1 μM TLR4 inhibitor (TAK 242) or DMSO prior to being seeded to mixtures of pFn (10 μg/ml) and EDA or the control module III-10n (40 μg/ml) in 0.1% BSA-DMEM for 2 hours. RNA was extracted and EDA+Fn and total Fn mRNA levels were assessed by real time RT-PCR analysis. Fold change values are expressed relative to NT siRNA treated cells plated to control wells. Error bars indicate standard error of quadruplicate samples for one of three representative experiments. Statistical analysis was performed using a two-way ANOVA with Tukey post-hoc test. **P<0.01, ***P<0.001 compared to IgG control or siRNA control.

Article Snippet: Blocking antibody to human TLR4 was obtained from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Blocking Assay, Quantitative RT-PCR, Transfection