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: Aging (Albany NY)

Article Title: TLR3 deletion inhibits programmed necrosis of brain cells in neonatal mice with sevoflurane-induced cognitive dysfunction

doi: 10.18632/aging.204092

Figure Lengend Snippet: TLR3 deletion inhibited activation of TRIF and degradation of SHP2 and down-regulated expressions of TLR signaling pathway-related proteins. ( A ) Expressions of TLR3, p-TRIF and SHP2 in the hippocampus of neonatal mice. ( B ) Expressions of p-RIP3 and CaMKII in the hippocampus of neonatal mice. ( C ) Expressions of NLRP3, GSDMD, Caspase-1, Caspase-4 and Caspase-11 in the hippocampus of neonatal mice. ** p < 0.01, and * p < 0.05.

Article Snippet: The hippocampal neurons were extracted from WT, TLR3 KO (6–8 weeks of age, purchased from Cyagen Biotechnology Co., Ltd.) and receptor-interacting protein 3 (RIP3) KO neonatal mice (Born within 24-hours) for in vitro experiments.

Techniques: Activation Assay

Journal: Aging (Albany NY)

Article Title: TLR3 deletion inhibits programmed necrosis of brain cells in neonatal mice with sevoflurane-induced cognitive dysfunction

doi: 10.18632/aging.204092

Figure Lengend Snippet: TLR3 deletion inhibited RIP3 phosphorylation and reduced programmed necrosis of hippocampal neurons by regulating TRIF and SHP2 expressions. ( A ) Expressions of TLR3, p-TRIF, SHP2, p-RIP3 and CaMKII in the hippocampal neurons. ( B ) Expressions of NLRP3, GSDMD, Caspase-1, Caspase-4 and Caspase-11 in the hippocampal neurons. ** p < 0.01.

Article Snippet: The hippocampal neurons were extracted from WT, TLR3 KO (6–8 weeks of age, purchased from Cyagen Biotechnology Co., Ltd.) and receptor-interacting protein 3 (RIP3) KO neonatal mice (Born within 24-hours) for in vitro experiments.

Techniques: Phospho-proteomics

Journal: Journal of Functional Foods

Article Title: Rutin mitigates hepatic fibrogenesis and inflammation through targeting TLR4 and P2X7 receptor signaling pathway in vitro and in vivo

doi: 10.1016/j.jff.2019.103700

Figure Lengend Snippet: Fig. 3. Rutin mediated TLR4 and P2X7r signaling pathways reverses the activation of HSCs (A) Representative western blotting analysis of TLR4, TLR2, TLR3, CD14, and MyD88. (B) Representative western blotting analysis of P2X7r and ASC. Representative RT-PCR analysis for expressions of P2X7r. (C) Representative western blotting analysis of IRAK1, IRAK4, TRAF-1, and IRF-3. Densitometric values were normalized against GAPDH. (D) Immunofluorescence staining of TLR2, TLR3, TLR4, P2X7r, and IRAK4 present in 400× magnification. *P < 0.05, **P < 0.01, ***P < 0.001 vs TGF-β group, ns, no significance.

Article Snippet: Primary antibodies of caspase-1 (sc-514), IL-1α (sc-393998), IL-1β (sc32294), IL-6 (sc-28343), ASC (sc-514414), TLR3 (sc-32232), TLR4 (sc293072), TRAF-1 (sc-7186), IRF-3 (sc-33641), and CD14 (sc-9150) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Protein-Protein interactions, Activation Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Journal of Functional Foods

Article Title: Rutin mitigates hepatic fibrogenesis and inflammation through targeting TLR4 and P2X7 receptor signaling pathway in vitro and in vivo

doi: 10.1016/j.jff.2019.103700

Figure Lengend Snippet: Fig. 8. Rutin ameliorates hepatic fibrosis by regulating TLR4 signaling in TAA-induced mice. (A) Representative western blotting analysis of TLR2, TLR3, TLR4, CD14, and MyD88. Densitometric values were normalized against GAPDH. (B) Immunofluorescence staining of TLR4 present in 600× magnification. (C) Representative western blotting analysis of IRAK1, IRAK4, TRAF-1, and IRF-3. Densitometric values were normalized against GAPDH. (D) Representative RT-PCR analysis for expressions of TLR4 and CD14. Densitometric values were normalized against GAPDH. (E) Immunofluorescence staining of IRAK1 and IRAK4 present in 200× magnification. ###P < 0.001 vs normal group, *P < 0.05, **P < 0.01, ***P < 0.001 vs TAA group, ns, no significance.

Article Snippet: Primary antibodies of caspase-1 (sc-514), IL-1α (sc-393998), IL-1β (sc32294), IL-6 (sc-28343), ASC (sc-514414), TLR3 (sc-32232), TLR4 (sc293072), TRAF-1 (sc-7186), IRF-3 (sc-33641), and CD14 (sc-9150) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Western Blot, Staining, Reverse Transcription Polymerase Chain Reaction

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: Cellular & Molecular Biology Letters

Article Title: IRE1α arm of unfolded protein response in muscle-specific TGF-β signaling-mediated regulation of muscle cell immunological properties

doi: 10.1186/s11658-023-00429-w

Figure Lengend Snippet: Intrinsic TGF-β signal controls immunological molecules and myokine expression in cultured primary myotubes. A Representative immunofluorescence staining for desmin, myogenin, eMHC, and TGF-βr2 in SM TGF-βr2 −/− mice-derived MPC-myotubes (MPCs) that received pro-inflammatory stimuli or not. B , C Western blot analysis of the protein expression of p-Smad2/3, H-2K b , H2-Eα, and TLR3 in MPC-myotubes that received pro-inflammatory stimuli or not. D Luminex assay of protein level changes for pro-inflammatory myokines in control or SM TGF-βr2 −/− mice-derived MPC-myotubes exposed to inflammatory milieu or not. Before creating the heat map, log transformation was used to process the data and plot in R language. The relative protein levels are expressed as a ratio (protein of interest/GAPDH). All data are presented as means ± SD ( n = 3 replicates). One-way ANOVA was used for multiple comparisons (** P < 0.01; *** P < 0.001). Bar = 50 μm

Article Snippet: The following primary antibodies were used: rabbit anti-mouse TGF-β2 (1: 500, Bioss, bs-20412R), rabbit anti-mouse TGF-βr2 (1: 500, Bioss, bs-0117R), mouse anti-mouse TLR3 (1:1000, NOVUS, NBP2-24875), rabbit anti-mouse H-2K b (1:500, Abcam, ab93364), mouse anti-mouse H2-Eα (1:1000, NOVUS, NBP1-43312), rabbit anti-mouse IRE1α (1:1000, NOVUS, NB100-2324), rabbit anti-mouse IRE1α (p Ser724) (1:1500, NOVUS, NB100-2323), mouse anti-mouse eIF2α (1:1000, Abcam, ab5369), rabbit anti-mouse eIF2α (p S51) (1:1000, Abcam, ab32157), mouse anti-mouse ATF6 (1:5000, NOVUS, NBP1-40256), rabbit anti-mouse p65 (1:1000, CST, 8242), rabbit anti-mouse P-p65 (1:1000, CST, 3033), rabbit anti-mouse Erk1/2 (1:1000, CST, 4695), rabbit anti-mouse P-Erk1/2 (1:1000, CST, 4370), mouse anti-mouse p38 (1:1000, Santa Cruz, sc-7972), mouse anti-mouse p38 (1:1000, Santa Cruz, sc-166182), rabbit anti-mouse JNK (1:1000, CST, 9252), rabbit anti-mouse P-JNK (1:1000, CST, 9255), rabbit anti-mouse Smad2/3 (1:500, Abcam, ab217553), and mouse anti-mouse GAPDH (1:5000, Fudebio, FD0063-100).

Techniques: Expressing, Cell Culture, Immunofluorescence, Staining, Derivative Assay, Western Blot, Luminex, Control, Transformation Assay

Journal: Cellular & Molecular Biology Letters

Article Title: IRE1α arm of unfolded protein response in muscle-specific TGF-β signaling-mediated regulation of muscle cell immunological properties

doi: 10.1186/s11658-023-00429-w

Figure Lengend Snippet: Intrinsic TGF-β signaling affects muscle cell immune behaviors by prompting UPR activity under pro-inflammatory conditions. The protein levels of IRE1α and eIF2α ( A ), H-2K b , H2-Eα, and TLR3 ( B ) in control or SM TGF-βr2 −/− mice-derived MPC-myotubes, with or without stimulation of IFN-γ/LPS, Tg, or TM, were analyzed by Western blot. The relative protein expression values are expressed as a ratio [protein of interest/GAPDH or phosphorylated (P) protein/total (T) protein]. C mRNA levels of IL-1β, IL-6, and MCP-1 in MPC-myotubes with or without 48 h stimulation of IFN-γ/LPS, Tg, or TM, analyzed by qPCR. All data are presented as means ± SD ( n = 3). A one-way ANOVA was used for multiple comparisons (* P < 0.05; ** P < 0.01)

Article Snippet: The following primary antibodies were used: rabbit anti-mouse TGF-β2 (1: 500, Bioss, bs-20412R), rabbit anti-mouse TGF-βr2 (1: 500, Bioss, bs-0117R), mouse anti-mouse TLR3 (1:1000, NOVUS, NBP2-24875), rabbit anti-mouse H-2K b (1:500, Abcam, ab93364), mouse anti-mouse H2-Eα (1:1000, NOVUS, NBP1-43312), rabbit anti-mouse IRE1α (1:1000, NOVUS, NB100-2324), rabbit anti-mouse IRE1α (p Ser724) (1:1500, NOVUS, NB100-2323), mouse anti-mouse eIF2α (1:1000, Abcam, ab5369), rabbit anti-mouse eIF2α (p S51) (1:1000, Abcam, ab32157), mouse anti-mouse ATF6 (1:5000, NOVUS, NBP1-40256), rabbit anti-mouse p65 (1:1000, CST, 8242), rabbit anti-mouse P-p65 (1:1000, CST, 3033), rabbit anti-mouse Erk1/2 (1:1000, CST, 4695), rabbit anti-mouse P-Erk1/2 (1:1000, CST, 4370), mouse anti-mouse p38 (1:1000, Santa Cruz, sc-7972), mouse anti-mouse p38 (1:1000, Santa Cruz, sc-166182), rabbit anti-mouse JNK (1:1000, CST, 9252), rabbit anti-mouse P-JNK (1:1000, CST, 9255), rabbit anti-mouse Smad2/3 (1:500, Abcam, ab217553), and mouse anti-mouse GAPDH (1:5000, Fudebio, FD0063-100).

Techniques: Activity Assay, Control, Derivative Assay, Western Blot, Expressing

Journal: Cellular & Molecular Biology Letters

Article Title: IRE1α arm of unfolded protein response in muscle-specific TGF-β signaling-mediated regulation of muscle cell immunological properties

doi: 10.1186/s11658-023-00429-w

Figure Lengend Snippet: TGF-β-IRE1α signaling inhibits immunological characteristics of muscle cells by attenuating p38 MAPK pathway. Western blot analyses of the expression changes of H-2K b , H2-Eα, or TLR3 in MPC-myotubes derived from Con and SM TGF-βr2 −/− mice ( A ), or in TGF-βr2 −/− MPC-myotubes ( B ) with or without stimulation of IFN-γ/LPS, SRI, 4μ8C, and/or SB (p38 pathway inhibitor). The relative protein expression values are expressed as a ratio (protein of interest/GAPDH). All data are presented as means ± SD ( n = 3). A one-way ANOVA was used for multiple comparisons (* P < 0.05; ** P < 0.01)

Article Snippet: The following primary antibodies were used: rabbit anti-mouse TGF-β2 (1: 500, Bioss, bs-20412R), rabbit anti-mouse TGF-βr2 (1: 500, Bioss, bs-0117R), mouse anti-mouse TLR3 (1:1000, NOVUS, NBP2-24875), rabbit anti-mouse H-2K b (1:500, Abcam, ab93364), mouse anti-mouse H2-Eα (1:1000, NOVUS, NBP1-43312), rabbit anti-mouse IRE1α (1:1000, NOVUS, NB100-2324), rabbit anti-mouse IRE1α (p Ser724) (1:1500, NOVUS, NB100-2323), mouse anti-mouse eIF2α (1:1000, Abcam, ab5369), rabbit anti-mouse eIF2α (p S51) (1:1000, Abcam, ab32157), mouse anti-mouse ATF6 (1:5000, NOVUS, NBP1-40256), rabbit anti-mouse p65 (1:1000, CST, 8242), rabbit anti-mouse P-p65 (1:1000, CST, 3033), rabbit anti-mouse Erk1/2 (1:1000, CST, 4695), rabbit anti-mouse P-Erk1/2 (1:1000, CST, 4370), mouse anti-mouse p38 (1:1000, Santa Cruz, sc-7972), mouse anti-mouse p38 (1:1000, Santa Cruz, sc-166182), rabbit anti-mouse JNK (1:1000, CST, 9252), rabbit anti-mouse P-JNK (1:1000, CST, 9255), rabbit anti-mouse Smad2/3 (1:500, Abcam, ab217553), and mouse anti-mouse GAPDH (1:5000, Fudebio, FD0063-100).

Techniques: Western Blot, Expressing, Derivative Assay