Journal: BMC Musculoskeletal Disorders

Article Title: TLR2 and IRF8 are the biomarkers associated with hypoxia in steroid-induced osteonecrosis of the femoral head via bioinformatics analysis and experimental validation

doi: 10.1186/s12891-025-09132-7

Figure Lengend Snippet: ROC curves analysis of 5 hub genes. ( A ) ROC curve of TLR2 in SONFH. ( B ) ROC curve of IRF8 in SONFH. ( C ) ROC curve of CXCL8 in SONFH. ( D ) ROC curve of CD86 in SONFH. ( E ) ROC curve of MAPK1 in SONFH.

Article Snippet: Human TLR2 ELISA kit (No. CSB-E11781h, Cusabio, Wuhan, China), human IRF8 ELISA kit (No. A- QEK00354 , Biogradetech, USA), and human CXCL8 ELISA kit (No. CSB-E04641h, Cusabio, Wuhan, China), human CD86 ELISA kit (No. CSB-E08542h, Cusabio, Wuhan, China), and human MAPK1 ELISA kit (No. A- QEK07595 , Biogradetech, USA) were used for ELISA according to the manufacturer’s instructions.

Techniques:

Journal: Nature Microbiology

Article Title: Gut–liver translocation of pathogen Klebsiella pneumoniae promotes hepatocellular carcinoma in mice

doi: 10.1038/s41564-024-01890-9

Figure Lengend Snippet: a , Representative SEM (top) and TEM (bottom) images of Hep3B and Huh7 HCC cell lines after co-culture with K. pneumoniae (MOI = 10). After co-culture of HCC cells with K. pneumoniae , cell lysate was spread on BHI agar for bacterial culture. n = 3 independent experiments with similar results. b , Effect of pasteurized K. pneumoniae (MOI = 10) on HCC cell proliferation (top) ( n = 3 biologically independent samples), colony formation (middle) ( n = 3 biologically independent samples) and growth of HCC patient-derived organoid (bottom) ( n = 20 biologically independent samples). c , Effect of pasteurized K. pneumoniae (MOI = 10) on PCNA expression in HCC cells. n = 3 (Ctrl), 6 (pasteurized KP). d , Screening of K. pneumoniae adhesins by biotin pull-down assay. e , Hep3B membrane protein was incubated with GST or GST–PBP1B together with GST magnetic beads for GST pull-down assay. Corresponding bands in GST–PBP1B and Hep3B cell membrane protein groups were subjected to mass spectrometry analysis. In d and e , n = 3 independent experiments with similar results. f , Binding affinity between PBP1B and TLR4 was detected using SPR. K d , dissociation constant. g , Representative structure of PBP1B and TLR4 after molecular docking (left) and the residues involved in the interaction between PBP1B with TLR4 (right). The structure of TLR4 is coloured pink while the PBP1B is coloured yellow. The light green dash represents hydrogen bond. h , TLR4 from Hep3B and Huh7 cell lysates was pulled down by GST–PBP1B according to the GST pull-down assay. i , Immunoprecipitation of TLR4 from Hep3B or Huh7 cells lysates validated the binding between TLR4 and GST–PBP1B. In h and i , n = 3 independent experiments with similar results. j , Effect of PBP1B (0.05 μM) on HCC cell proliferation (top) ( n = 3 biologically independent samples), colony formation (middle) ( n = 3 biologically independent samples) and growth of HCC patient-derived organoid (bottom) ( n = 20 biologically independent samples). n = 3 independent experiments with similar results. k , Effect of PBP1B (0.05 μM) on TLR4, MyD88, p-P65, P65 and PCNA protein expression in HCC cells, as determined by western blot. n = 3 biologically independent samples. l , m , TLR4i (30 μM) abolished the effect of PBP1B on HCC cell proliferation ( l ) and colony formation ( m ). n = 3 biologically independent samples. n , Effect of K. pneumoniae on TLR4 expression in liver tissues of both germ-free and SPF mice with or without DEN treatment by IHC staining. In b , c , j – m , data are presented as mean ± s.e.m. Each data point in bar plots represents one mouse. Cell proliferation was analysed using two-way ANOVA. In b , c , j , k , m , comparisons between two groups were analysed using Student’s t -test. Unless otherwise stated, statistical significance was calculated using one-way ANOVA. Adjustments were made for multiple comparisons.

Article Snippet: TLR4 human recombinant protein (HY-P73586, MCE) was immobilized on a Series S Sensor Chip CM5 (29149603, GE Healthcare) using an amine coupling kit (BR-1000-50, GE Healthcare) with a flow rate of 10 μl min −1 , resulting in a final ligand coupling of ~180 response units.

Techniques: Co-Culture Assay, Derivative Assay, Expressing, Pull Down Assay, Membrane, Incubation, Magnetic Beads, Mass Spectrometry, Binding Assay, Immunoprecipitation, Western Blot, Immunohistochemistry

Journal: Nature Microbiology

Article Title: Gut–liver translocation of pathogen Klebsiella pneumoniae promotes hepatocellular carcinoma in mice

doi: 10.1038/s41564-024-01890-9

Figure Lengend Snippet: (a) Design of K. oxytoca on K. pneumoniae induced HCC promotion in germ-free mice with DEN treatment (GFD/PBS group n=6; GFD/EC group n=6; GFD/KO group n=6; GFD/KP group n=7; GFD/KOP group n=8) and relevant results (b–e) . (b) Images of liver gross morphology (yellow circles indicate tumor area), and H&E with tumor incidence, tumor number and tumor burden. n=6 (GFD/PBS), 6 (GFD/EC), 6 (GFD/KO) 7 (GFD/KP), 8 (GFD/KOP). (c) Alcian blue staining, E-Cad, CLDN 3, CLDN 1 IHC, and Cy3-conjugated EUB338 probe detection with quantitative analysis. n=6 biologically independent samples. (d) Gut permeability assay using FITC-dextran. n=6 (GFD/PBS), 6 (GFD/EC), 6 (GFD/KO) 7 (GFD/KP), 8 (GFD/KOP). (e) Live bacteria culture of liver tissues on blood agar plates with quantitative analysis. n=6 (GFD/PBS), 6 (GFD/EC), 6 (GFD/KO) 7 (GFD/KP), 8 (GFD/KOP). (f) Design of TLR4i on K. pneumoniae induced HCC promotion in germ-free mice with DEN treatment (GFD/PBS group n=6; GFD/EC group n=6; GFD/KP group n=7; GFD/KPi group n=7) and relevant results (g-k) . (g) Images of liver gross morphology (yellow circles indicate tumor area), and H&E with tumor incidence, tumor number and tumor burden. n=6 (GFD/PBS), 6 (GFD/EC), 7 (GFD/KP), 7 (GFD/KPi). (h) E-Cad IHC and Cy3-conjugated EUB338 probe (GFD/PBS group), E. coli probe (GFD/EC), K. pneumoniae probe (GFD/KP) and GFD/KPi groups) detection of colon tissues with quantitative analysis. n=6 biologically independent samples. (i) Gut permeability assay using FITC-dextran. n=6 biologically independent samples. (j) Live bacteria culture of liver tissues on blood agar plates with quantitative analysis. n=6 (GFD/PBS), 6 (GFD/EC), 7 (GFD/KP), 7 (GFD/KPi). (k) Cy3-conjugated conjugated EUB338 probe (GFD/PBS group), E. coli probe (GFD/EC), K. pneumoniae probe (GFD/KP) and GFD/KPi groups) FISH, TLR4, PCNA, Ki-67 IHC, and Sirius red staining of liver sections. n=6 biologically independent samples. Data (excluding tumor incidence and liver with live bacteria) are presented as mean ± SEM. Each data point in bar plots represents one mouse. Tumor incidence and Liver with live bacteria were calculated using Fisher’s exact test. Unless otherwise stated, statistical significance was calculated using one-way ANOVA. Adjustments were made for multiple comparisons.

Article Snippet: TLR4 human recombinant protein (HY-P73586, MCE) was immobilized on a Series S Sensor Chip CM5 (29149603, GE Healthcare) using an amine coupling kit (BR-1000-50, GE Healthcare) with a flow rate of 10 μl min −1 , resulting in a final ligand coupling of ~180 response units.

Techniques: Staining, Permeability, Bacteria

Journal: Nature Microbiology

Article Title: Gut–liver translocation of pathogen Klebsiella pneumoniae promotes hepatocellular carcinoma in mice

doi: 10.1038/s41564-024-01890-9

Figure Lengend Snippet: (a) Design of TLR4i on HCC-FMT induced HCC promotion in germ-free mice with DEN treatment (GFD/PBS group n=8; GFD/HCC-FMT group n=15; GFD/HCC-FMTi group n=10) and relevant results (b–g) . TLR4i was daily gavaged in this experiment. (b) Representative images of liver gross morphology (yellow circles indicate tumor area), and H&E staining of mice liver sections with tumor incidence, tumor number, and tumor burden. n=8 (GFD/PBS), 15 (GFD/HCC-FMT), 10 (GFD/HCC-FMTi). (c) Representative bacterial culture of liver tissues under anaerobic and aerobic conditions with quantitative analysis. n=8 (GFD/PBS), 15 (GFD/HCC-FMT), 10 (GFD/HCC-FMTi). From left to right and top to bottom, the culture plates are blood agar plate, chocolate blood agar plate, MacConkey agar plate, and Columbia blood agar plate, respectively. Gut permeability assay using 500 kDa FITC-dextran. n=6 biologically independent samples. (d) Representative pictures of Alcian blue staining, E-Cad IHC staining, and Cy3-conjugated EUB338 probe FISH detection of colon tissues with quantitative analysis. n=6 biologically independent samples. (e) Cy3-conjugated EUB338 probe FISH, Cy3-conjugated K. pneumoniae probe FISH, TLR4, PCNA, Ki-67 IHC of liver sections. n=6 biologically independent samples. (f) α-SMA, Desmin IHC staining, and Sirius red staining of liver sections. n=6 biologically independent samples. (g) Mouse Cancer Pathway Finder Array and Inflammatory Response and Autoimmunity PCR Array of liver tissues. n=3 independent experiments with similar results. Data (excluding tumor incidence, liver with live bacteria and PCR array results) are presented as mean ± SEM. Each data point in bar plots represents one mouse. Tumor incidence and Liver with live bacteria were calculated using Fisher’s exact test. Unless otherwise stated, statistical significance was calculated using one-way ANOVA. Adjustments were made for multiple comparisons.

Article Snippet: TLR4 human recombinant protein (HY-P73586, MCE) was immobilized on a Series S Sensor Chip CM5 (29149603, GE Healthcare) using an amine coupling kit (BR-1000-50, GE Healthcare) with a flow rate of 10 μl min −1 , resulting in a final ligand coupling of ~180 response units.

Techniques: Staining, Chocolate, Permeability, Immunohistochemistry, Bacteria

Journal: Virchows Archiv : an international journal of pathology

Article Title: Toll-like receptors 1, 2, 4, 5, and 6 in gastric cancer.

doi: 10.1007/s00428-023-03635-1

Figure Lengend Snippet: Fig. 1 Representative images of cytoplasmic TLR2 and TLR6 expression immunostaining in gastric adenocarcinoma. Low cytoplasmic TLR2 expression (A), high cytoplasmic TLR2 expression (B), low cytoplasmic TLR6 expression (C), and high cytoplasmic TLR6 expression (D)

Article Snippet: Samples were rinsed in distilled water and phosphate-buffered saline with Tween (PBS-T) and endogenous peroxidase was then neutralized in peroxidase blocking solution (Dako S2023) for 5 min. After a wash in PBS-T, sections were incubated with antibodies (Dako S2022); TLR1 (diluted 1:300, ROCKLAND 800-656-7625), TLR2 (diluted 1:500, ROCKLAND 600-401-956), TLR4 (diluted 1:1000, abonova 214-291), TLR5 (1:75, NOVUS NBP-2-24787), and TLR6 (diluted 1:750, abonova PAB 3555).

Techniques: Expressing, Immunostaining

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Advanced Glycation End-Product of Low Density Lipoprotein Activates the Toll-Like 4 Receptor Pathway Implications for Diabetic Atherosclerosis

doi: 10.1161/atvbaha.108.175992

Figure Lengend Snippet: Figure 2. Wild-type (TLR4/) and TLR4 knock-out (TLR4 /) macrophages were treated as indicated and culture medium assayed for IL6. Probability values 0.05, comparing treatments to unstimulated control, are asterisked. Probability values 0.05, comparing 24-hour cytokine expression values between the same stimulus in TLR4/ and TLR4/ macrophages, are shown by a cross (†). n4.

Article Snippet: Full length wild-type TLR4 (Origene, Cambridge Bioscience) was subcloned into the plasmid vector pcDNA4-His-Max-C (Invitrogen). pGL3-basic-MMP1 plasmid has been described previously.19 The IL8 promoter (2kb) was generated by polymerase chain reaction (PCR) using genomic DNA as template and cloned into pGL3-basic (Promega).

Techniques: Knock-Out, Control, Expressing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Advanced Glycation End-Product of Low Density Lipoprotein Activates the Toll-Like 4 Receptor Pathway Implications for Diabetic Atherosclerosis

doi: 10.1161/atvbaha.108.175992

Figure Lengend Snippet: Figure 4. AGE-LDL activates kinases through TLR4. HEK293-MD2-CD14, transfected with empty vector or TLR4 receptor, were incu- bated with AGE-LDL for the specified times. Total cell extracts were assayed for phospho-p38, phospho-JNK [pan], phospho-ERK1, and phospho-Akt S473 [pan]. t tests were performed against the unstimulated-no-TLR4 control at zero hours. probability values 0.05 are asterisked. n4.

Article Snippet: Full length wild-type TLR4 (Origene, Cambridge Bioscience) was subcloned into the plasmid vector pcDNA4-His-Max-C (Invitrogen). pGL3-basic-MMP1 plasmid has been described previously.19 The IL8 promoter (2kb) was generated by polymerase chain reaction (PCR) using genomic DNA as template and cloned into pGL3-basic (Promega).

Techniques: Transfection, Plasmid Preparation, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Advanced Glycation End-Product of Low Density Lipoprotein Activates the Toll-Like 4 Receptor Pathway Implications for Diabetic Atherosclerosis

doi: 10.1161/atvbaha.108.175992

Figure Lengend Snippet: Figure 5. AGE-LDL activates transcription factors via TLR4. HEK293-MD2-CD14, transfected with empty vector or the TLR4 receptor, were incubated with AGE-LDL. Nuclear extracts were assayed for p-Jun, Elk-1, or NFB. t tests were performed against the unstimulated-no-TLR4 control at zero hours. Differ- ences with a probability value 0.05 are asterisked. n4 to 6.

Article Snippet: Full length wild-type TLR4 (Origene, Cambridge Bioscience) was subcloned into the plasmid vector pcDNA4-His-Max-C (Invitrogen). pGL3-basic-MMP1 plasmid has been described previously.19 The IL8 promoter (2kb) was generated by polymerase chain reaction (PCR) using genomic DNA as template and cloned into pGL3-basic (Promega).

Techniques: Transfection, Plasmid Preparation, Incubation, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Advanced Glycation End-Product of Low Density Lipoprotein Activates the Toll-Like 4 Receptor Pathway Implications for Diabetic Atherosclerosis

doi: 10.1161/atvbaha.108.175992

Figure Lengend Snippet: Figure 6. AGE-LDL promotes cytokine synthesis. a, HEK293-MD2-CD14 transfected with empty vector or the TLR4 plasmid were incu- bated with AGE-LDL. After 24 hours the culture medium was assayed for IL6 or TNF. t tests comparing AGE-LDL treated cells with unstimulated cells were performed. Differences with a probability value 0.05 are asterisked. n5.

Article Snippet: Full length wild-type TLR4 (Origene, Cambridge Bioscience) was subcloned into the plasmid vector pcDNA4-His-Max-C (Invitrogen). pGL3-basic-MMP1 plasmid has been described previously.19 The IL8 promoter (2kb) was generated by polymerase chain reaction (PCR) using genomic DNA as template and cloned into pGL3-basic (Promega).

Techniques: Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E 2 and 15-deoxy-Δ 12,14 -PGJ 2

doi: 10.1371/journal.pone.0027630

Figure Lengend Snippet: T/C-28a2 chondrocytes were treated with either PGD 2 (A) or 15d-PGJ 2 (B) for 48 h, or PGE 2 (C) for 2 h. TLR4, caveolin-1 and IL-6 protein (upper) and mRNA (lower) expression was determined by Western blotting or qRT-PCR, respectively. ß-actin and GAPDH served as internal controls in immunoblotting and qRT-PCR, respectively. The Western blots are representative of three independent experiments, all revealing similar results. Data represent the mean ± S.E. of 3 independent qRT-PCR experiments. * and ▴, p <0.05 with respect to the corresponding vehicle control.

Article Snippet: The caveolin-1 and TLR4 cDNA plasmids were supplied from Origene Technologies (Rockville, MD), and subcloned to the pCMV6-XL vector.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control

Journal: PLoS ONE

Article Title: Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E 2 and 15-deoxy-Δ 12,14 -PGJ 2

doi: 10.1371/journal.pone.0027630

Figure Lengend Snippet: T/C-28a2 cells were incubated with either PGE 2 (10 µM) for 2 h, or PGD 2 (8 µM) or 15d-PGJ 2 (1 µM) for 48 h. In other experiments, T/C-28a2 cells were pre-treated with PGD 2 (8 µM) or 15d-PGJ 2 (1 µM) for 48 h before incubation with PGE 2 (10 µM) for 2 h. In select experiments, T/C-28a2 cells were transfected with a siRNA oligonucleotide sequence specific for TLR4 (B, C) or caveolin-1 (D) or a plasmid containing the cDNA of TLR4 (D) or caveolin-1 (B, C) before PG treatment. TLR4 (A, C, D), caveolin-1 (A, C, D) and IL-6 (A, C, D) mRNA synthesis was determined by qRT-PCR. GAPDH served as internal control. Data represent the mean ± S.E. of at least 3 independent experiments. *, p <0.05 with respect to vehicle or mock transfected control. ▴, p <0.05 with respect to significantly regulated (*) groups. IL-6 (B) is shown by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of ß-actin. These western blots are representative of three independent experiments, all revealing similar results.

Article Snippet: The caveolin-1 and TLR4 cDNA plasmids were supplied from Origene Technologies (Rockville, MD), and subcloned to the pCMV6-XL vector.

Techniques: Incubation, Transfection, Sequencing, Plasmid Preparation, Quantitative RT-PCR, Control, Western Blot

Journal: PLoS ONE

Article Title: Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E 2 and 15-deoxy-Δ 12,14 -PGJ 2

doi: 10.1371/journal.pone.0027630

Figure Lengend Snippet: T/C-28a2 cells were transfected with an siRNA oligonucleotide sequence specific for TLR4 (A) or caveolin-1 (B). In select experiments, T/C-28a2 cells were transfected with a plasmid containing the cDNA of TLR4 (A) or caveolin-1 (B). In separate experiments, cells were incubated with PGE 2 (10 µM) for 2 h in the presence or absence of the MEK1/2 inhibitors, PD98059 (20 µM) or U0126 (10 µM) (C). cAMP accumulation (A, B) was determined using a cAMP enzyme immunoassay kit. IL-6 mRNA synthesis (C right panel) was determined by qRT-PCR. GAPDH served as internal control. Data represent the mean ± S.E. of at least 3 independent experiments. *, p <0.05 with respect to scramble siRNA or empty vector transfected control. ▴, p <0.05 with respect to significantly regulated (*) groups. IL-6 and phosphorylated ERK1/2 (Thr202/Tyr204) (C left panel) are shown by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of total ERK1/2 and ß-actin. These western blots are representative of three independent experiments, all revealing similar results.

Article Snippet: The caveolin-1 and TLR4 cDNA plasmids were supplied from Origene Technologies (Rockville, MD), and subcloned to the pCMV6-XL vector.

Techniques: Transfection, Sequencing, Plasmid Preparation, Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Western Blot

Journal: PLoS ONE

Article Title: Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E 2 and 15-deoxy-Δ 12,14 -PGJ 2

doi: 10.1371/journal.pone.0027630

Figure Lengend Snippet: T/C-28a2 cells were incubated with either PGE 2 (10 µM) for 2 h (A) or 15d-PGJ 2 (1 µM) for 48 h (C, D). In other experiments, cells were pre-treated with 15d-PGJ 2 (1 µM) for 48 h before incubation with PGE 2 (10 µM) for 2 h (B). In select experiments, cells were transfected with an siRNA oligonucleotide sequence specific for TLR4 (B) or caveolin-1 (D) or a plasmid containing the cDNA of caveolin-1 (B) or TLR4 (D) before treatment with PGE 2 or 15d-PGJ 2 . Phosphorylated ERK1/2 (Thr202/Tyr204), Akt (Ser 473) and CREB (Ser 133) are shown by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of total ERK1/2, Akt, CREB and ß-actin. TLR4 and caveolin-1 protein levels were also probed with an anti-TLR4 and an anti-caveolin-1 antibody, respectively. These western blots are representative of three independent experiments, all revealing similar results.

Article Snippet: The caveolin-1 and TLR4 cDNA plasmids were supplied from Origene Technologies (Rockville, MD), and subcloned to the pCMV6-XL vector.

Techniques: Incubation, Transfection, Sequencing, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E 2 and 15-deoxy-Δ 12,14 -PGJ 2

doi: 10.1371/journal.pone.0027630

Figure Lengend Snippet: T/C-28a2 cells were transfected with an siRNA oligonucleotide sequence specific for TLR4 (A) or caveolin-1 (B) or a plasmid containing the cDNA of caveolin-1 (A) or TLR4 (B) before treatment with PGE 2 (10 µM) for 2 h (A) or 15d-PGJ 2 (1 µM) for 48 h (B). In select experiments, cells were pretreated with 15d-PGJ 2 (1 µM) for 48 h before incubation with PGE 2 (10 µM) for 2 h (A). Phosphorylated p65 (Ser536 and Ser276) is detected by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of total p65 and ß-actin. These Western blots are representative of three independent experiments, all revealing similar results.

Article Snippet: The caveolin-1 and TLR4 cDNA plasmids were supplied from Origene Technologies (Rockville, MD), and subcloned to the pCMV6-XL vector.

Techniques: Transfection, Sequencing, Plasmid Preparation, Incubation, Western Blot

Journal: PLoS ONE

Article Title: Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E 2 and 15-deoxy-Δ 12,14 -PGJ 2

doi: 10.1371/journal.pone.0027630

Figure Lengend Snippet: T/C-28a2 cells were pre-treated with 15d-PGJ 2 (1 µM) for 48 h before incubation with PGE 2 (10 µM) for 2 h (A). In other experiments, cells were incubated with either PGE 2 (10 µM) for 2 h (B, C, F) or 15d-PGJ 2 (1 µM) for 48 h (D, E). In select experiments, T/C-28a2 cells were transfected with a plasmid containing an siRNA oligonucleotide sequence specific for TLR4 (B) or caveolin-1 (E) or the cDNA of caveolin-1 (C) or TLR4 (D) before PG treatment. In separate experiments, cells were incubated with PGE 2 for 2 h in the presence or absence of the MEK1/2 inhibitors, PD98059 (20 µM) or U0126 (10 µM) (F). Cells were transfected with the indicated siRNAs or cDNA constructs along with the IL-6 promoter reporter construct pIL-6-luc651 or pIL-6-luc651 ΔNF-κB before PG stimulation, as described under “Experimental Procedures”. Luciferase activities were measured by using the Dual-Luciferase Reported Assay kit and normalized to sea pansy luciferase activity of co-transfected pRL-SV40. Data represent the mean ± S.E. of at least 3 independent experiments. *, p <0.05 with respect to the pIL-6-luc651 ΔNF-κB and vehicle or mock transfected control. ▴, p <0.05 with respect to significantly regulated (*) groups.

Article Snippet: The caveolin-1 and TLR4 cDNA plasmids were supplied from Origene Technologies (Rockville, MD), and subcloned to the pCMV6-XL vector.

Techniques: Incubation, Transfection, Plasmid Preparation, Sequencing, Construct, Luciferase, Activity Assay, Control

Journal: PLoS ONE

Article Title: Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E 2 and 15-deoxy-Δ 12,14 -PGJ 2

doi: 10.1371/journal.pone.0027630

Figure Lengend Snippet: T/C-28a2 cells were incubated with either PGE 2 (10 µM) for 2 h or 15d-PGJ 2 (1 µM) for 48 h. In other experiments, cells were pre-treated with 15d-PGJ 2 (1 µM) for 48 h before incubation with PGE 2 (10 µM) for 2 h. In select experiments, T/C-28a2 cells were transfected with an siRNA oligonucleotide sequence specific for TLR4 (A, B) or caveolin-1 (C, D) or a plasmid containing the cDNA of caveolin-1 (A, B) or TLR4 (C, D) before PG treatment. In separate experiments, cells were incubated with PGE 2 for 2 h in the presence or absence of the MEK1/2 inhibitors, PD98059 (20 µM) or U0126 (10 µM) (E, F). Nuclear extracts were then isolated, and NF-κB-specific DNA-protein complex formation was determined by gel shift (A, C, E). Supershift assays (B, D, F) using an anti-p65 Ab were carried out as outlined in “Experimental Procedures”. Results of a competition experiment using 200-fold unlabeled NF-κB oligonucleotide (cold probe) are shown. These gels are representative of three independent experiments, all revealing similar results.

Article Snippet: The caveolin-1 and TLR4 cDNA plasmids were supplied from Origene Technologies (Rockville, MD), and subcloned to the pCMV6-XL vector.

Techniques: Incubation, Transfection, Sequencing, Plasmid Preparation, Isolation, Gel Shift

Journal: PLoS ONE

Article Title: Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E 2 and 15-deoxy-Δ 12,14 -PGJ 2

doi: 10.1371/journal.pone.0027630

Figure Lengend Snippet: T/C-28a2 cells were incubated with either PGE 2 (10 µM) for 2 h or 15d-PGJ 2 (1 µM) for 48 h. In other experiments, cells were pre-treated with 15d-PGJ 2 (1 µM) for 48 h before incubation with PGE 2 (10 µM) for 2 h (A). In select experiments, T/C-28a2 cells were transfected with an siRNA oligonucleotide sequence specific for TLR4 (B) or caveolin-1 (C) or a plasmid containing the cDNA of caveolin-1 (B) or TLR4 (C) before PG treatment. In separate experiments, cells were incubated with PGE 2 for 2 h in the presence or absence of the MEK1/2 inhibitors, PD98059 (20 µM) or U0126 (10 µM) (D). Crosslinked chromatin was immunoprecipitated using an anti-p65 antibody. In ChIP assays, the anti-RNA polymerase II antibody was used as positive control, whereas the normal mouse IgG and anti-TLR4 antibodies were used as negative controls. DNA purified from both the immunoprecipitated (IP) and pre-immune ( input ) specimens were subjected to qRT-PCR amplification using primers for the GAPDH (control) and p65 promoter genes. Data represent the mean ± S.E. of at least 3 independent experiments. *, p <0.05 with respect to scramble siRNA or empty vector transfected control. ▴, p <0.05 with respect to significantly regulated (*) groups.

Article Snippet: The caveolin-1 and TLR4 cDNA plasmids were supplied from Origene Technologies (Rockville, MD), and subcloned to the pCMV6-XL vector.

Techniques: Incubation, Transfection, Sequencing, Plasmid Preparation, Immunoprecipitation, Positive Control, Purification, Quantitative RT-PCR, Amplification, Control

Journal: PLoS ONE

Article Title: Interleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E 2 and 15-deoxy-Δ 12,14 -PGJ 2

doi: 10.1371/journal.pone.0027630

Figure Lengend Snippet: PGE 2 stimulates TLR4 synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-κB p65 subunit leading to NF-κB activation. Binding of the activated NF-κB p65 subunit to IL-6 promoter induces IL-6 synthesis in human chondrocytes. PGD 2 or 15d-PGJ 2 concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE 2 -dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-κB-dependent IL-6 synthesis in chondrocytes.

Article Snippet: The caveolin-1 and TLR4 cDNA plasmids were supplied from Origene Technologies (Rockville, MD), and subcloned to the pCMV6-XL vector.

Techniques: Activation Assay, Binding Assay

Journal: Reviews in Cardiovascular Medicine

Article Title: Toll-Like Receptor 4 Is an Early and Sensitive Biomarker to Detect Acute Kidney Injury after Surgery for Type A Aortic Dissection

doi: 10.31083/j.rcm2311363

Figure Lengend Snippet: Toll-like receptor 4 levels (mean and standard deviation) of patients in the AKI group and the non-AKI group at multiple time points .

Article Snippet: The level of TLR4 was determined by a human TLR4 ELISA kit (RK02399, ABclonal, Nanjing, China), according to the manufacturer’s instruction.

Techniques: Standard Deviation

Journal: Reviews in Cardiovascular Medicine

Article Title: Toll-Like Receptor 4 Is an Early and Sensitive Biomarker to Detect Acute Kidney Injury after Surgery for Type A Aortic Dissection

doi: 10.31083/j.rcm2311363

Figure Lengend Snippet: Receiver operating characteristic (ROC) curves before surgery, 0-hour, 1-hour, 3-hour, 6-hour, 12-hour, and 24-hour toll-like receptor 4 levels after ICU stay in predicting acute kidney injury (AKI) after acute type A aortic dissection (ATAAD) surgery . AUC, area under the curve; CI, confidence interval.

Article Snippet: The level of TLR4 was determined by a human TLR4 ELISA kit (RK02399, ABclonal, Nanjing, China), according to the manufacturer’s instruction.

Techniques: Dissection

Journal: Reviews in Cardiovascular Medicine

Article Title: Toll-Like Receptor 4 Is an Early and Sensitive Biomarker to Detect Acute Kidney Injury after Surgery for Type A Aortic Dissection

doi: 10.31083/j.rcm2311363

Figure Lengend Snippet: Receiver operating characteristic (ROC) curve comparing prior to surgery, 0-hour, 1-hour, 3-hour, 6-hour, 12-hour, and 24-hour toll-like receptor 4 levels after ICU stay in predicting acute kidney injury (AKI) (stage 3 AKI) after acute type A aortic dissection (ATAAD) surgery . AUC, area under the curve; CI, confidence interval.

Article Snippet: The level of TLR4 was determined by a human TLR4 ELISA kit (RK02399, ABclonal, Nanjing, China), according to the manufacturer’s instruction.

Techniques: Dissection

Journal: Reviews in Cardiovascular Medicine

Article Title: Toll-Like Receptor 4 Is an Early and Sensitive Biomarker to Detect Acute Kidney Injury after Surgery for Type A Aortic Dissection

doi: 10.31083/j.rcm2311363

Figure Lengend Snippet: Toll-like receptor 4 levels (mean and standard deviation) of patients stratified with 30-day mortality at multiple time points .

Article Snippet: The level of TLR4 was determined by a human TLR4 ELISA kit (RK02399, ABclonal, Nanjing, China), according to the manufacturer’s instruction.

Techniques: Standard Deviation

Journal: Reviews in Cardiovascular Medicine

Article Title: Toll-Like Receptor 4 Is an Early and Sensitive Biomarker to Detect Acute Kidney Injury after Surgery for Type A Aortic Dissection

doi: 10.31083/j.rcm2311363

Figure Lengend Snippet: Receiver operating characteristic (ROC) curve comparing the ability before surgery, 0-hour, 1-hour, 3-hour, 6-hour, 12-hour, and 24-hour toll-like receptor 4 levels after ICU stay in predicting 30-day mortality after acute type A aortic dissection (ATAAD) surgery . AUC, area under the curve; CI, confidence interval.

Article Snippet: The level of TLR4 was determined by a human TLR4 ELISA kit (RK02399, ABclonal, Nanjing, China), according to the manufacturer’s instruction.

Techniques: Dissection