Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR3 activation augments matrix metalloproteinase production through reactive nitrogen species generation in human lung fibroblasts.

doi: 10.4049/jimmunol.1302919

Figure Lengend Snippet: FIGURE 9. Responsiveness of NHLFs and DHLF-As to poly(I:C). NHLFs (normal) (n = 3) and DHLF-As (asthma) (n = 4) were incubated with 30 mg/ml poly(I:C) or vehicle for 48 h. MMP-9 (A and B), -2 (A and C) and -1 (D) were analyzed by zymography. Fold increase was calculated by dividing the band intensity of MMPs in the poly(I:C)-treated group by that in the vehicle-treated group. The fold increases in the MMPs from NHLFs are compared with those from DHLF-As (B–D). Expression of TLR3 (E) and iNOS (F) in the cells was analyzed by immunoblotting. *p , 0.05 versus NHLFs. M, markers of MMPs.

Article Snippet: The cells were incubated with mouse monoclonal anti-TLR3 Ab (1:100 dilution; Imgenex) or mouse IgG as a negative control for detection of TLR3.

Techniques: Incubation, Zymography, Expressing, Western Blot

Journal: Journal of Histochemistry and Cytochemistry

Article Title: The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer

doi: 10.1369/0022155416656154

Figure Lengend Snippet: Used TLR Antibodies With Dilutions, Catalog Numbers, and Manufacturer.

Article Snippet: TLR3 , 1:25 , IMG-315A , Imgenex.

Techniques:

Journal: Journal of Histochemistry and Cytochemistry

Article Title: The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer

doi: 10.1369/0022155416656154

Figure Lengend Snippet: Typical Toll-like receptor (TLR) expression patterns from the human ascending colon. TLRs are expressed throughout the epithelium with a diffuse manner. Paired figures from ascending colon organ donor (left) and tumor-adjacent normal epithelium (right) are presented: (A) TLR1, (B) TLR2, (C) TLR3, (D) TLR4, (E) TLR5, (F) TLR6, (G) TLR7, (H) TLR8, and (I) TLR9. 20× magnification was used and 50-µm scale bar is in panel I.

Article Snippet: TLR3 , 1:25 , IMG-315A , Imgenex.

Techniques: Expressing

Journal: Journal of Histochemistry and Cytochemistry

Article Title: The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer

doi: 10.1369/0022155416656154

Figure Lengend Snippet: TLR1 to TLR9 Protein Expression Detected With Immunohistochemistry From Different Anatomic Segments of the Alimentary Tract.

Article Snippet: TLR3 , 1:25 , IMG-315A , Imgenex.

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Histochemistry and Cytochemistry

Article Title: The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer

doi: 10.1369/0022155416656154

Figure Lengend Snippet: Typical Toll-like receptor (TLR) expression patterns from the conventional and germ-free mouse small intestines. TLRs are expressed throughout the epithelium with a diffuse manner. Paired figures from small intestine conventional (left) and germ-free mice (right) are presented: (A) TLR1, (B) TLR2, (C) TLR3, (D) TLR4, (E) TLR5, (F) TLR6, (G) TLR7, (H) TLR8, and (I) TLR9. 10× magnification was used and 50-µm scale bar is in panel I.

Article Snippet: TLR3 , 1:25 , IMG-315A , Imgenex.

Techniques: Expressing

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: A and B, TLR3 colocalized with SREC-I in HEK 293-TLR3-SREC-I overexpressing cells. HEK293 cells were transfected with FLAG-SREC-I and TLR3-CFP for 22 hours. Cells were then incubated without (A) or with (B) Poly I:C (10ug/ml) on ice for 30 min. Cells (A) and (B) were then fixed with 4% para formaldehyde and permeabilized with 0.1% Triton X100 (A) or not (B). Cells were then stained for FLAG with anti-FLAG M2 antibody (green) C, HEK293 cells were transfected with FLAG-SREC-I and TLR3-CFP for 22 hr. Cells were then incubated Poly I:C (10ug/ml) on ice for 30 min followed by incubation with warm media at 37°C for 20 min. Cells were then fixed with 4% PFA and permeabilized with 0.1% Triton X100. Cells were stained with anti-FLAG antibody (green). D, HEK293 cells were transfected with FLAG-SREC-1 or TLR3-CFP for 18 hours. Cells were incubated with Poly I:C (10μg/ml) for 1 hour and then fixed and stained with anti-FLAG M2 ab (green). E, Differentiated THP1 cells were transfected with FLAG-SREC-I and TLR3-CFP for 22 hr. Cells were then incubated with Poly I:C (10μg/ml) for 1 hr and then fixed and permeabilized as in D. Cells were stained for SREC-I (green), with anti FLAG ab, LAMP1 with anti LAMP1 (red). TLR3-CFP is in blue. Experiments were repeated twice with reproducible findings. Scale bar 2 micron.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Transfection, Incubation, Staining

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: HEK293 cells stably expressing TLR3 was transfected with SREC-I-GFP for 22 hr. Cells were then treated with or without 10ug/ml Poly I:C for 30 min. Cell lysates were collected and SREC-I-GFP was immunoprecipitated using anti-GFP antibody and anti GFP ab and then the precipitated complexes were subjected to SDS-PAGE analysis followed by blotting for TLR3 using anti-TLR3 antibody. Experiments were carried out twice, reproducibly.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Stable Transfection, Expressing, Transfection, Immunoprecipitation, SDS Page

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: A, B and C, HEK293-TLR3 cells were transfected with SREC-I or untransfected HEK293-TLR3 cells were then incubated with or without Poly I:C (10μg/ml) for 2 hr. Cell lysates were collected and then subjected to SDS-PAGE and western blotting with appropriate antibodies. D, Bone marrow derived macrophages (BMDM) were transfected with siRNA of SREC-I for 72 hr. Cells were then incubated with Poly I:C (or not) as in A. Cell were lysed and equal amount of protein were subjected to SDS-PAGE and western blotting using anti phospho-p65 antibody and anti p65 antibody. E, BMDM cells were treated as in D and then cell lysates were subjected to SDS-PAGE and western blotting using antibodies shown in E. F, HEK293-TLR3 cells were transfected with SREC-I or not and also NFkβ-SEAP/CMV-SEAP constructs. Cells were incubated with Poly I:C (10μg/ml)/ODN2395 (10μg/ml), a non TLR3 ligand. NFkβ activity was measured as instructed by NFkβ-SEAporter assay kit. Similar results were observed in two separate experiments.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Transfection, Incubation, SDS Page, Western Blot, Derivative Assay, Construct, Activity Assay

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: A, THP1 cells were transfected with TLR3 or TLR3 and SREC-I expression plasmids for 22 hr. Cells were incubated with 10ug Poly I:C for 12 hr and then assayed for cytokine production using a human cytokine multianylate ELISA array kit according to manufacturer’s protocol. B, THP1 cells treated as in (A) and then assayed for chemokines using multianylate ELISA array kit according to Manufacturer’s protocol. Data represent the mean of two independent experiments.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Transfection, Expressing, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: A, THP1 cells were transfected with TLR3/SREC-I or TLR3 and SREC-I expression plasmids for 22 hr. Cells were incubated with Poly I:C (10ug/ml) for 12 hr and then assayed for IL-8 release. B, THP1 cells were transfected with SREC-I and TLR3 for 22 hr and then incubated with the ERK inhibitor (PD98059), p38 inhibitor (SB203580) or the JNK inhibitor (JNK inhibitor II) (10uM) for 1 hr right before incubation with incubation with Poly I:C for 12 hr. IL-8 secretion was assayed according to manufacturer’s instructions. C, THP1 cells were treated as in A and then IL-6 release was assayed according to manufacturer’s instruction. D, THP1 cells were treated as in B and IL-6 release was assayed. E, THP1 cells were treated with PMA (5–10ng/ml) for 72 hours. Cells were then transfected with siRNA SREC-I or siRNA TLR3. Cells were incubated with Poly I:C for 12 hr and then Ifnβ release was measured according to manufacturer’s protocol. F. BMDM cells were transfected with siRNA SREC-I/TLR3 or not. Wild type or TLR3 knocked down (siRNA) cells were also treated with 1–5 ng/ml of LPS for 12hrs. Cells were then incubated with Poly I:C for 12 hr. Secreted IL-6 in media was measured according to manufacturer’s protocol. Experiment was repeated twice. Data shown are the mean ±SD of results from those two experiments.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Transfection, Expressing, Incubation

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: A, THP1-TLR3 cells were transfected with or without SREC-I for 22 hr and then incubated with 10ug Poly I:C for indicated time. IL-8 release from cells was then assayed. B, THP1-TLR3 cells were transfected as in A and then incubated with or without Bafilomycin A (0.2 uM for 20 min) or PP2 (10uM for 12 hr). Cells were incubated with Poly I:C for 12 hr and then IL-8 release from THP1 cells were assayed. C, Differentiated THP1 cells were transfected with siRNA SREC-I for 72 hr and then cells were treated with Poly I:C (10μg/ml) for 2 hr. Cells were then lysed and equal amount of protein in lysate were subjected to SDS-PAGE and western blotting using appropriate antibodies. Experiments were carried out twice reproducibly.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Transfection, Incubation, SDS Page, Western Blot

Journal: Immunobiology

Article Title: Scavenger receptor SREC-I promotes double stranded RNA-mediated TLR3 activation in human monocytes

doi: 10.1016/j.imbio.2014.12.011

Figure Lengend Snippet: Double stranded RNA species Poly I:C interacts with macrophages leading to recruitment of SREC-I and TLR3 to the cell surface where they form a membrane complex that interacts with c-Src. The latter kinase then regulates endocytosis of the SREC-I/TLR3/Poly I:C complexes in endosomes. The complex finally resides in endosomes with low intravesicular pH, marked with lysosomal protein LAMP1. TLR3 is able to signal from such complexes and launches NFkB, MAPK and IRF3 signaling. (IRF3 is activated by the kinase TBK1). Activated NFkB, AP-1 and C/EBPβ are known to interact with the IL-8 gene while activated IRF3 leads to synthesis of Ifn-β.

Article Snippet: Both human and mice SREC-I (siRNA) and TLR3 (siRNA) constructs were purchased from Santa Cruz Biotech.

Techniques: Membrane

Journal: PLoS Pathogens

Article Title: Class A Scavenger Receptor 1 (MSR1) Restricts Hepatitis C Virus Replication by Mediating Toll-like Receptor 3 Recognition of Viral RNAs Produced in Neighboring Cells

doi: 10.1371/journal.ppat.1003345

Figure Lengend Snippet: ( A ) Quantitative RT-PCR analysis of (left) IFN-β and (center and right) ISG56 mRNA in Huh7.5-TLR3 cells infected with HCV. Cells were infected with HCV (HJ3-5) or UV-inactivated HCV at an m.o.i. of 1 for 72 hrs, prior to extraction of RNA and measurement of IFN-β and ISG56 mRNA. Statistical comparisons were made between mock- and HCV-infected cultures (left and center panels), or between samples indicated by bars (right panel): * p≤0.02, ** p≤0.001. ( B ) Agarose gel electrophoresis of in vitro transcribed HCV RNAs (top panel) before or (bottom panel) after S1 nuclease treatment. M: DNA standards ladder; lane 1, positive-strand HCV RNA; lane 2, negative-strand HCV RNA; lane 3, annealed double-stranded HCV RNA. ( C ) Quantitative RT-PCR analysis of ISG56 mRNA in (left) PH5CH8 and (right) Huh7.5/TLR3 cells treated with 50 µg/ml of ssRNA or dsRNA for 6 hrs prior to extraction of RNA. Statistical comparisons were made with cultures not treated with RNA: * p≤0.02, ** p≤0.001. ( D ) (left panel) Immunoblot analysis of ISG15 expression induced by synthetic HCV dsRNA in PH5CH8 cells previously transduced with lentivirus vectors expressing non-targeting (NT) or TLR3-specific shRNAs. β-actin was included as a loading control. (right panel) Similar immunoblots of Huh7.5-TLR3 cells treated with HCV dsRNA, either with or without prior S1 nuclease digestion. In both panels, cell lysates were prepared for immunoblotting 24 hrs after addition of dsRNA. ( E ) HCV RNA co-immunoprecipitates with Flag-TLR3 from lysates of HCV-infected Huh7.5-TLR3 cells. (top panel) Agarose gel electrophoresis showing detection of HCV-specific RNA by RT-PCR in anti-Flag (lanes 2, 4 and 6), or isotype control (mouse IgG 1 , lanes 1, 3 and 5), immunoprecipitates prepared from lysates of Huh7.5-TLR3 cells (lanes 1 and 2), Huh7.5-H539E cells (lanes 3 and 4), or Huh7.5-H539E cells (lanes 5 and 6), infected with HJ3-5 virus (m.o.i. = 1) for 72 hrs. RNA extracted from mock or HCV-infected Huh-7.5 cells was assayed in parallel in the RT-PCR reaction as negative and positive controls, respectively (lanes 7 and 8). (bottom panel) Anti-Flag immunoblots of the respective immunoprecipitates.

Article Snippet: 293-hTLR3 cells (engineered to over-express human TLR3) were purchased from InvivoGen.

Techniques: Quantitative RT-PCR, Infection, Extraction, Agarose Gel Electrophoresis, In Vitro, Western Blot, Expressing, Transduction, Reverse Transcription Polymerase Chain Reaction, Virus

Journal: PLoS Pathogens

Article Title: Class A Scavenger Receptor 1 (MSR1) Restricts Hepatitis C Virus Replication by Mediating Toll-like Receptor 3 Recognition of Viral RNAs Produced in Neighboring Cells

doi: 10.1371/journal.ppat.1003345

Figure Lengend Snippet: ( A ) Immunoblot analysis of MSR1 expression in (top panels) Huh7.5-TLR3 and (bottom panels) PH5CH8 cells transduced with MSR1-specific (shMSR1) or non-targeting control shRNA (shNT) lentiviruses. ( B ) Effect of shRNA-mediated MSR1 depletion on uptake of FITC-labeled poly(I:C) by Huh7.5-TLR3 or PH5CH8 cells. Cells were incubated with 10 µg/ml FITC-labeled poly(I:C) for 8, 16 or 24 hrs before harvest. Cells were washed extensively before fixation and analyzed by flow cytometry. Uptake of FITC-labeled poly(I:C) is expressed as fold-change in mean fluorescence intensity (MFI) compared to mock-treated cells. Statistical comparisons between the MFI at 8 hrs vs. the time indicated were made by two-way ANOVA with Bonferroni correction for multiple comparisons: * p≤0.02, ** p≤0.001. ( C ) Effect of shRNA-mediated MSR1 depletion on TLR3 signaling. IFN-β and PRD-II promoter activation was induced by extracellular poly(I:C) in MSR1-depleted (left) Huh7.5-TLR3 and (right) PH5CH8 cells exposed to 50 µg/ml of high molecular weight poly(I:C) for 6 hrs. Statistical comparisons in this and other panels in this figure were between the shNT and shMSR1-treated cells: * p≤0.02, ** p≤0.001. ( D ) Quantitative RT-PCR analysis of ISG56 mRNA in MSR1-depleted cells treated with poly(I:C) as in panel C. ( E ) Immunoblots of ISG15 expression in MSR1-depleted (left) Huh7.5-TLR3 and (right) PH5CH8 cells treated with poly(I:C) for 24 hrs. ( F ) Immunoblot of ISG15 in MSR1-depleted Huh7.5-TLR3 cells exposed to synthetic HCV dsRNA 50 µg/ml for 24 hrs. ( G ) Quantitative RT-PCR analysis of ISG56 mRNA in Huh7.5-TLR3/shMSR1 or Huh7.5-TLR3/shNT cells infected with HJ3-5 virus at an m.o.i. of 1 for 72 hrs. ( H ) Viral replication assessed by the fold-change in secreted Gaussia luciferase in cultures of Huh7.5-TLR3/shMSR1 vs. Huh7.5-TLR3/shNT cells following infection with HJ3-5/GLuc2A virus. The level of Gaussia luciferase activity at each time point was calculated relative to that at 6 hrs after infection, which was set at 1. Data are the mean ± s.d. from three independent experiments. The difference in GLuc expression by Huh7.5-TLR3/shMSR1 vs. Huh7.5-TLR3/shNT cells was significant by two-way ANOVA (p = 0.006). Bonferroni post-tests were used to estimate the significance of differences at individual time points: * p≤0.02. ( I ) Quantitative RT-PCR analysis of HCV RNA in Huh7.5-TLR3/shMSR1 vs. Huh7.5-TLR3/shNT cells 72 hrs after infection with HCV. RNA abundance in the Huh7.5-TLR3/shMSR1 was calculated relative to that in Huh7.5-TLR3/shNT, which was set at 100%. ( J ) Co-immunoprecipitation of HCV RNA with Flag-TLR3 in lysates of Huh7.5-TLR3/shMSR1 (lanes 3, 4, and 6) and Huh7.5-TLR3/shNT cells (lanes 1, 2 and 5) infected with HJ3-5 virus 72 hrs previously. Immunoprecipitation was with anti-Flag. See legend to for additional details.

Article Snippet: 293-hTLR3 cells (engineered to over-express human TLR3) were purchased from InvivoGen.

Techniques: Western Blot, Expressing, Transduction, shRNA, Labeling, Incubation, Flow Cytometry, Fluorescence, Activation Assay, Molecular Weight, Quantitative RT-PCR, Infection, Virus, Luciferase, Activity Assay, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Class A Scavenger Receptor 1 (MSR1) Restricts Hepatitis C Virus Replication by Mediating Toll-like Receptor 3 Recognition of Viral RNAs Produced in Neighboring Cells

doi: 10.1371/journal.ppat.1003345

Figure Lengend Snippet: ( A ) Reconstitution of MSR1 expression in Huh7.5-TLR3/shMSR1 cells. Myc-tagged MSR1 was stably expressed by retroviral transfer of a shMSR1-resistant vector (pCX4bsr/Myc-MSR1). Lysates were immunoprecipitated with anti-Myc, then subjected to immunoblotting as described in . “Vector” = cells transduced with empty vector. ( B ) Restoration of poly(I:C) induction of IFN-β promoter activity by ectopically expressed Myc-MSR1 in Huh7.5-TLR3/shMSR1 cells. Cells were co-transfected with pCX4bsr/Myc-MSR1 (vs. empty vector), pIFN-β-Luc and pRL-CMV (internal control reporter) and cultured for 24 h, then treated with poly(I:C) (50 µg/ml) for 6 hrs prior to lysis and luciferase assay. Huh7.5-TLR3/shNT cells, transfected with empty vector, were included as a positive control. ( C ) Quantitative RT-PCR analysis of HCV RNA in Huh7.5-TLR3/shMSR1 cells stably expressing Myc-MSR1 (vs. empty vector) 72 hrs after infection with HJ3-5 virus at an m.o.i. of 1. ( D ) Co-immunoprecipitation of HCV RNA with Myc-MSR1 in lysates of Huh7.5-TLR3/shMSR1 cells stably expressing Myc-MSR1 (lanes 3 and 6) vs. empty vector (lanes 2 and 5). Cells were infected with HJ3-5 virus (m.o.i. = 1) 72 hrs prior to lysis. Immunoprecipitation was with anti-Myc, demonstrating association of HCV RNA with Myc-MSR1. See legend to for further details.

Article Snippet: 293-hTLR3 cells (engineered to over-express human TLR3) were purchased from InvivoGen.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Transduction, Activity Assay, Transfection, Cell Culture, Lysis, Luciferase, Positive Control, Quantitative RT-PCR, Infection, Virus

Journal: PLoS Pathogens

Article Title: Class A Scavenger Receptor 1 (MSR1) Restricts Hepatitis C Virus Replication by Mediating Toll-like Receptor 3 Recognition of Viral RNAs Produced in Neighboring Cells

doi: 10.1371/journal.ppat.1003345

Figure Lengend Snippet: ( A ) (top) Domain architecture of the 451 a.a. human MSR1 protein showing the location of the collagen superfamily domain that contains multiple G-X-Y repeats that form a triple helix . (bottom) Alignment of human MSR1 with other mammalian MSR1 proteins near the C-terminus of the collagen domain, showing multiple conserved basic residues. GenBank accession numbers are shown for each. “Macscav” = conserved macrophage scavenger receptor domain; “SRCR” = Cys-rich scavenger receptor superfamily domain. ( B ) Myc-MSR1 mutants constructed within the pCX4bsr/Myc-MSR1 plasmid. ( C ) Poly(I:C) stimulated IFN-β promoter activity in MSR1-depleted Huh7.5 TLR3/shMSR1 cells transiently expressing wt or mutant Myc-MSR1: (left) Myc-MSR1/Δ321–339 (deletion of underlined sequence in panel A), (center) Myc-MSR1/3KA, or (right) Myc-MSR1/R325A and Muc-MSR1/R3KA. Cells were co-transfected with Myc-MSR1 expression vectors, pIFN-β-Luc and pRL-CMV (internal control), cultured for 24 h, then treated with poly(I:C) (50 µg/ml) for 6 hrs before to lysis and luciferase assay. ( D ) Immunoblots showing expression of wt Myc-MSR1 and related mutants in PH5CH8/shMSR1 cells. Lysates were precipitated with anti-Myc antibody prior to anti-Myc immunoblot. ( E ) Flow cytometry histograms showing expression of Myc-MSR1 (wt) and indicated mutants on the surface of PH5CH8 cells (green lines). Purple curves indicate cell-surface staining with isotype immunoglobulins. “FL2-H” = fluorescent intensity. ( F ) Co-immunoprecipitation analysis of the association of HCV RNA with wt versus mutant Myc-MSR1 proteins in PH5CH8/shMSR1 cells co-cultured with Huh7.5 cells infected with HJ3-5 virus (m.o.i. = 1) for 72 hrs. RNA was extracted anti-Myc precipitates from lysates of co-cultures of infected Huh7.5 cells and PH5CH8/shMSR1 cells stably transduced with empty vector (lane 1 and 5), wt Myc-MSR1 (lane 2 and 6), Δ321–339 (lane 3 and 7), or R3KA (lane 4 and 8). See legend to for further details.

Article Snippet: 293-hTLR3 cells (engineered to over-express human TLR3) were purchased from InvivoGen.

Techniques: Construct, Plasmid Preparation, Activity Assay, Expressing, Mutagenesis, Sequencing, Transfection, Cell Culture, Lysis, Luciferase, Western Blot, Flow Cytometry, Staining, Immunoprecipitation, Infection, Virus, Stable Transfection, Transduction

Journal: PLoS Pathogens

Article Title: Class A Scavenger Receptor 1 (MSR1) Restricts Hepatitis C Virus Replication by Mediating Toll-like Receptor 3 Recognition of Viral RNAs Produced in Neighboring Cells

doi: 10.1371/journal.ppat.1003345

Figure Lengend Snippet: ( A ) Experimental design, showing co-culture of HCV-nonpermissive, TLR3-competent 293FT/IFN-β-mCherry cells expressing mCherry under control of the IFN-β promoter with Huh-7.5 cells that are HCV permissive and TLR3 incompetent. Cells were infected with HJ3-5/5A-YFP virus that expresses YFP as a fusion with NS5A. ( B ) Immunofluorescence microscopy demonstrating induction of mCherry expression in 293FT/IFN-β-mCherry + Huh-7.5 cell co-cultures upon stimulation with poly(I:C) or infection with HJ3-5/NS5A-YFP virus. HCV replication was visualized by YFP expression and present in cells adjacent to those expressing mCherry in the two-color merged images at the far right. Nuclei were visualized by DAPI counterstain. ( C ) siRNA-mediated depletion of TLR3 significantly reduces poly-(I:C)-induced activation of the IFN-β promoter in 293FT/IFN-β-mCherry cells. ( left ) Fold-change in luciferase activity induced by extracellular poly-(I:C) in 293FT/IFN-β-mCherry cells transfected with TLR3-specific (siTLR3) or control (siCont) siRNAs. The cells were transduced with an IFN-β-Luc reporter plasmid. * p≤0.02. ( right ) qRT-PCR detection of TLR3-specific mRNA in cells transfected with the siTLR3 or siCont siRNAs. * p≤0.02. ( D ) Poly-(I:C)-induced expression of mCherry in 293FT/IFN-β-mCherry cells is ablated by prior transfection with TLR3-specific siRNA. ( E ) siRNA-mediated depletion of TLR3 eliminates mCherry expression by 293FT/IFN-β-mCherry cells placed in co-culture with HCV-infected Huh-7.5 cells. 293FT/IFN-β-mCherry cells were transfected with siTLR3 or siCont siRNAs 3 days prior to being placed in co-culture with Huh-7.5 cells infected with genotype 2a JFH-1 virus. ( F ) Experimental design, showing Huh7.5-TLR3 cells co-cultured with Huh7.5 cells supporting replication of a genome-length genotype 1a HCV RNA replicon. ( G ) Co-immunoprecipitation analysis of the association of HCV RNA with Flag-TLR3 in co-cultures of Huh7.5-TLR3 and HCV replicon cells (panel F) after treatment with bafilomycin (1.0 nM, lanes 2 and 5) or chloroquine (5.0 µM, lanes 3 and 6) for 72 hrs. RNA was extracted from anti-Flag precipitates prepared from lysates of the co-cultured cells and subjected to HCV-specific RT-PCR. See legend to for further details.

Article Snippet: 293-hTLR3 cells (engineered to over-express human TLR3) were purchased from InvivoGen.

Techniques: Co-Culture Assay, Expressing, Infection, Virus, Immunofluorescence, Microscopy, Activation Assay, Luciferase, Activity Assay, Transfection, Transduction, Plasmid Preparation, Quantitative RT-PCR, Cell Culture, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: Class A Scavenger Receptor 1 (MSR1) Restricts Hepatitis C Virus Replication by Mediating Toll-like Receptor 3 Recognition of Viral RNAs Produced in Neighboring Cells

doi: 10.1371/journal.ppat.1003345

Figure Lengend Snippet: ( A ) ( left ) Design of co-culture experiments in which HCV-nonpermissive, but TLR3-competent, PH5CH8 cells were co-cultured with HCV-permissive, but RIG-I and TLR3-incompetent, Huh-7.5 cells infected with HJ3-5/GLuc2A, a reporter virus that expresses GLuc as a component of its polyprotein. Huh-7.5 cells were infected with virus for 6 hrs prior to being split and added to either MSR1-depleted PH5CH8/shMSR1 or control PH5CH8/shNT cells at a ratio of 1∶2 to establish co-cultures. ( right ) Gaussia luciferase activity in supernatant fluids of HJ3-5/GLuc2A-infected co-cultures. Data shown are means ± s.d. from 3 replicate cultures and are representative of repeat, independent experiments. The difference in GLuc expression from shMSR1- vs. shNT-treated cells was significant by two-way ANOVA (p<0.0001). ( B ) ( left ) Co-cultures of PH5CH8/shMSRI (or control PH5CH8/shNT) cells and HJ3-5/GLuc2A virus-infected Huh-7.5 cells in which the cell types are separated by a semi-permeable membrane (Transwell system). ( right ) Gaussia luciferase activity in supernatant fluids of HJ3-5/GLuc2A virus-infected Huh-7.5 cells separated from the PH5CH8 cells by a semi-permeable membrane. Data shown are means ± s.d. from 3 replicate cultures. Unlike Huh-7.5 cells cultured in close continuity with PH5CH8 cells (panel A), there is no restriction to HCV replication when the cell types are separated by a permeable membrane. The difference in GLuc expression from shMSR1- vs. shNT-treated cells was not significant by two-way ANOVA (p = 0.20).

Article Snippet: 293-hTLR3 cells (engineered to over-express human TLR3) were purchased from InvivoGen.

Techniques: Co-Culture Assay, Cell Culture, Infection, Virus, Luciferase, Activity Assay, Expressing, Membrane

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Figure Lengend Snippet: a Interferon [IFN]-β luciferase expression in HEK293T mSTING cells overexpressing cGAS treated O/N with the indicated compounds ( n = 3 independent experiments). b IFN-stimulated response element [ISRE]-Luciferase expression in L929 cells after 7 h stimulation with the mouse STING agonist DMXAA at 20 μg/ml and the indicated compounds ( n = 3 independent experiment for all except Quercetin n = 2 independent experiments). c IFN-β (right) and IP-10 (left) protein levels in THP-1 monocytes after 6 h stimulation with the human STING agonist GSK3 at 100 nM and treatment with indicated idronoxil (IDX) doses. Right: 2.5 μM IDX was used ( n = 3 independent experiments). d RNA sequencing analysis of the top 20 murine ISGs in RNA lysates from Trex1 Q98X bone marrow-derived macrophages [BMDMs] treated O/N with 1.25 μM IDX (heat map of log 2 expression relative to the average across all samples; significant genes are indicated with an asterisk) ( n = 3 independent animals [C1–C3]). NT is non-treated. e Immunoblot of wild-type [WT] immortalised bone marrow-derived macrophages [iBMDMs] stimulated with 50 μg/ml DMXAA (STING) for the indicated times ±2.5 μM IDX (1 representative blot of 3 independent experiments shown). Molecular weight markers are shown in kDa. A doublet of STING bands appears upon STING activation. Phosphorylated proteins are indicated with [P]. f Immunoblot of wild-type and Tbk1 KO iBMDMs stimulated with 200 ng/ml LPS (TLR4) ± 2.5 μM IDX treatment for the indicated time (1 representative blot of 3 independent experiments shown). Phosphorylated proteins are indicated with [P]. Molecular weight markers are shown in kDa. g IFN-β luciferase expression in p125HEK cells treated with indicated dose of IDX and stimulated O/N with 50 ng/ml of 3p-hpRNA (RIG-I agonist [RIG-Ia]) ( n = 3 independent experiments). h RT-qPCR analyses of IFNB1/18S, RSAD2/18S and IFIT1/18S in RNA lysates from primary human bronchial epithelial cells [PBECs] from 3 independent donors treated with 5 μM IDX or 200 nM MRT, and stimulated 3 h with 1 μg/ml transfected polyI:C (MDA5/RIG-I), 25 μg/ml naked polyI:C (TLR3) or transfected with 2.5 μg/ml ISD70 (cGAS) ( n = 3 independent donors). a – c , g , h Data are mean ± s.e.m. c One-way or ( h ) two-way ANOVA with uncorrected Fisher’s LSD (with single pooled variance) multiple comparisons are shown. a – c , g Non-linear regression analyses are shown. Exact p values are shown for all comparisons. Source data and detailed statistical analyses are provided as a Source Data file.

Article Snippet: The TLR4 agonist, LPS (#LPS-EB) and TLR3/RIG-I/MDA5 agonist high molecular weight (HMW) polyI:C (#tlrl-pic), and the tri-phosphate RNA selective RIG-I agonist (#3p-hpRNA) were from Invivogen.

Techniques: Luciferase, Expressing, RNA Sequencing Assay, Derivative Assay, Western Blot, Molecular Weight, Activation Assay, Quantitative RT-PCR, Transfection

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Figure Lengend Snippet: a Interferon [IFN]-β luciferase expression in HEK293T cells expressing the indicated constructs after O/N incubation ( n = 3 independent experiments). b Surface plasmon resonance [SPR] analysis of idronoxil (IDX) binding to recombinant human TBK1 (representative of n = 3 independent experiments). c qPCR amplification signal of kinase concentration after KinomeScan TM assays, for the indicated IDX concentrations (averaged from n = 2 technical replicates). IKKe is IKKε. d In silico modelling of IDX binding (bottom—green) and STING C-terminal tail (top—yellow, PDB: 6nt9) to TBK1 dimer (the blue and red residues refer to the two different TBK1 units in the structure). Immunoblot of Sting KO immortalised bone marrow-derived macrophages [iBMDMs] expressing mCitrine-STING, after 1 h stimulation with 50 μg/ml DMXAA (STING) and increasing doses of IDX ( e ), or 5 μM IDX or 100 nM MRT67307 [MRT] ( f ). e 1 representative blot of three independent experiments shown. f One representative blot of two independent experiments shown. Pink: pull-down of mCitrine-STING with anti-GFP antibody. Black: whole cell lysates. e , f Phosphorylated proteins are indicated with [P]. Molecular weight markers are shown in kDa. g IP-10 protein levels in wild-type (IC 50 = 1.6 μM), Tbk1 KO (IC 50 = 0.75 μM), Ikbkε KO [ Ikk ε KO ](IC 50 = 2 μM) and wild-type [WT] iBMDM cells after O/N stimulation with 50 μg/ml DMXAA (STING) and increasing doses of IDX ( n = 3 independent experiments). h HEK-TLR3 cells expressing NF-κB and IFN-β luciferase reporters were treated 6 h with 0.5 μg/ml poly(I:C) [pIC] (TLR3) in the presence of 5 μM IDX or 400 nM MRT ( n = 3 independent experiments). a , g , h Data are mean ± s.e.m. a – c , g Non-linear regression analyses are shown. h Two-way ANOVA with uncorrected Fisher’s LSD (with single pooled variance) multiple comparisons are shown. Exact p values are shown for all comparisons. Source data and detailed statistical analyses are provided as a Source Data file.

Article Snippet: The TLR4 agonist, LPS (#LPS-EB) and TLR3/RIG-I/MDA5 agonist high molecular weight (HMW) polyI:C (#tlrl-pic), and the tri-phosphate RNA selective RIG-I agonist (#3p-hpRNA) were from Invivogen.

Techniques: Luciferase, Expressing, Construct, Incubation, SPR Assay, Binding Assay, Recombinant, Amplification, Concentration Assay, In Silico, Western Blot, Derivative Assay, Molecular Weight