Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: Primer used for Real-time qPCR.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Sequencing

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: Cell surface level of TLR4 in BV-2 cells treated with chestnut leaf or spiny bur extracts and LPS. BV-2 cells were pre-treated (or not) with spiny bur (SB) or leaf (L) extracts (0.5 mg/mL) for 3 h, incubated with or without LPS (0.5 μg/mL) for a total of 24 h, then subjected to flow cytometric analysis of the immunostaining for TLR4 receptor. ( a ) Representative plots of untreated BV-2 cells ( b ) Representative plots of BV-2 cells not activated with LPS ( c ) Representative plots of LPS-stimulated BV-2 cells ( d ) Relative quantification is expressed as MFI, median fluorescence intensity. Results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. ° p < 0.05 significantly different from control cells; * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Incubation, Immunostaining, Fluorescence, Control

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: RT-PCR analysis of TLR4 gene expression in BV-2 cells treated with chestnut extracts. BV-2 cells were treated with spiny bur or leaf extracts (0.5 mg/mL) for 3 h, then stimulated (or not) with LPS (0.5 µg/mL) for a total of 24 h. At the end of incubation, RNA was extracted from cells and samples were subjected to RT-PCR analysis using a specific primer for TLR4, as explained in the Materials and Methods section. The relative mRNA content of TLR4 was normalised to ( a ) untreated control and ( b ) untreated LPS control. Relative quantification is obtained as reported in the Materials and Methods section; results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. ° p < 0.05 significantly different from control cells; * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Control

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: RT-PCR analysis of TLR4 and CD14 gene expression in BV-2 cells treated with different chestnut leaf extract fractions. BV-2 cells were treated for 3 h with 50 μg/mL of fractions at different polarity (L Fr.), then stimulated with 0.5 μg/mL LPS for a total of 24 h. At the end of incubation, RNA was extracted from cells and samples were subjected to RT-PCR analysis using a specific primer for TLR4 ( a ) and CD14 ( b ), as reported in the Materials and Methods section. The relative mRNA contents of TLR4 and CD14 were normalised to LPS-treated cells (red bars). Relative quantification is obtained as described in the Materials and Methods section, and results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 2. Upregulation of HMGB1, TLR4, pIKBα and GFAP in astrocytes and brain microvessel endothelial cells in MCAO rats. (A, B and D) Compared with the sham group, the expression of HMGB1, TLR4, pIKBα and GFAP was potentiated in the cerebral cortex tissues of MCAO rats. The scale bars represent 60 and 30 μm in (A) and (B) respectively. (C and E) Consistently, the HMGB1 (red) in astrocytes in MCAO group translocated from nucleus (Blue color represents DAPI) to the cytoplasm (Green color represents GFAP). The expression of TLR4 (yellow) and pIKBα (yellow) in brain microvessel endothelial cells (Green color represents VIII factor) in MCAO group was potentiated respectively. The expression of GFAP (green) in astrocytes (Blue color represents DAPI) in MCAO group was also increased. The scale bars represent 10 μm. (D and E) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques: Expressing

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 8. HMGB1 contributed to upregulation of P-gp via TLR4/NF-kB pathway. (A) The relationship among HMGB1 and changes of TLR4, TIRAP, NF-kB and P-gp in rBMECs after OGD was investigated using related positive agents. (B) Changes of TLR4 (green) and TIRAP (red) after OGD and treatment with positive agents in rBMECs. (C) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group; #P < 0.05 vs. OGD group. The scale bars represent 10 μm.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques:

Journal: Microbiome

Article Title: Ochratoxin A induces liver inflammation: involvement of intestinal microbiota

doi: 10.1186/s40168-019-0761-z

Figure Lengend Snippet: OTA promotes liver inflammation. a Relative mRNA expressions of TLR4, MYD88, IKBα, IL-6, and TNF-α in the liver after OTA oral gavage ( n = 6, mean with SEM). b Relative protein abundances of TLR4, MYD88, p-IKBα, p-IKBα/IKBα, and p-p65 in the liver after OTA oral gavage ( n = 6, mean with SEM). c Effect of OTA on levels of liver cytokines, including IL-1β, IL-6, TNF-α, IL-8, IL-10, and IFN-γ ( n = 6, mean with SEM). d Representative images of H&E-stained liver sections in CON and OTA group (magnification × 400, scale bar 100 μm, n = 6). e Statistical analysis of the percentage of inflammatory cells in different groups shown in d ( n = 6, mean with SEM). f Effect of OTA on serum levels of AST, ALT, ALP, and LDH ( n = 6, mean with SEM). g Serum LPS level with or without OTA treatment ( n = 6, mean with SEM). h Effect of OTA on serum levels of IL-1β, IL-6, TNF-α, and IL-10 ( n = 6, mean with SEM). Data in a , b , c , e , f , g , and h were analyzed with unpaired t test, * P < 0.05; ** P < 0.01, *** P < 0.001, P <0.0001

Article Snippet: Proteins were loaded onto the SDS-PAGE gel (BioRad) and electrophoresed and analyzed by WB using antibodies against TLR4 (BA1717, Boster, Wuhan, China), MYD88 (abs135682, Absin, Shanghai, China), IKBα (D120138, Sangon Biotech, Shanghai, China), p-IKBα (D151548, Sangon Biotech, Shanghai, China), p-p65 (HZ4902812, TW reagent, Shanghai, China), TJP-1 (mAb13663, Cell Signaling, USA), Occludin (ab167161, Abcam, USA), and actin (60008, Proteintech, USA).

Techniques: Staining

Journal: Microbiome

Article Title: Ochratoxin A induces liver inflammation: involvement of intestinal microbiota

doi: 10.1186/s40168-019-0761-z

Figure Lengend Snippet: OTA fails to promote liver inflammation in antibiotics-treated ducks. a Liver LPS level in different groups ( n = 6, mean with SEM). b Relative mRNA expressions of TLR4, MYD88, IKBα, IL-6, and TNF-α in the liver of antibiotics-treated ducks with or without OTA ( n = 6, mean with SEM). c Levels of IL-1β, IL-6, TNF-α, IL-8, IL-10, and IFN-γ in the liver of antibiotics-treated ducks with or without OTA ( n = 6, mean with SEM). d Representative images of H&E-stained liver sections (magnification × 400; scale bar 100 μm; n = 6). e Statistical analysis of the percentage of inflammatory cells in different groups shown in d ( n = 6, mean with SEM). f Effect of OTA on serum levels of AST, ALT, ALP and LDH in antibiotics-treated ducks ( n = 6, mean with SEM). g Serum LPS level with or without OTA treatment in antibiotics-treated ducks ( n = 6, mean with SEM). h Effect of OTA on serum levels of IL-1β, IL-6, TNF-α, and IL-10 ( n = 6, mean with SEM). Data were analyzed with unpaired t test. n.s., not significant

Article Snippet: Proteins were loaded onto the SDS-PAGE gel (BioRad) and electrophoresed and analyzed by WB using antibodies against TLR4 (BA1717, Boster, Wuhan, China), MYD88 (abs135682, Absin, Shanghai, China), IKBα (D120138, Sangon Biotech, Shanghai, China), p-IKBα (D151548, Sangon Biotech, Shanghai, China), p-p65 (HZ4902812, TW reagent, Shanghai, China), TJP-1 (mAb13663, Cell Signaling, USA), Occludin (ab167161, Abcam, USA), and actin (60008, Proteintech, USA).

Techniques: Staining

Journal: Microbiome

Article Title: Ochratoxin A induces liver inflammation: involvement of intestinal microbiota

doi: 10.1186/s40168-019-0761-z

Figure Lengend Snippet: OTA-originated microbiota induces liver inflammation. a Liver LPS level in FMT (CON) and FMT (OTA) ducks. b Relative mRNA expressions of TLR4, MYD88, IKBα, IL-6, and TNF-α in the liver after FMT ( n = 6, mean with SEM). c Relative protein abundance of TLR4, MYD88, p-IKBα, p-IKBα/IKBα, and p-p65 in the liver after FMT ( n = 6, mean with SEM). d Effect of FMT on the liver levels of IL-1β, IL-6, TNF-α, and IL-10 ( n = 6, mean with SEM). e Representative H&E-stained liver sections. f Statistical analysis of the percentage of inflammatory cells in different groups shown in e ( n = 6, mean with SEM). g Serum levels of AST, ALT, ALP, and LDH in different groups ( n = 6, mean with SEM). h Serum LPS level in different FMT groups ( n = 6, mean with SEM). i Serum levels of IL-1β, IL-6, and TNF-α after FMT ( n = 6, mean with SEM). FMT (CON): ducks received the CON group fecal microbiota. FMT (OTA): ducks received the OTA group fecal microbiota. Data were analyzed with unpaired t test, n = 6. * P < 0.05; ** P < 0.01, *** P < 0.001, **** P <0.0001

Article Snippet: Proteins were loaded onto the SDS-PAGE gel (BioRad) and electrophoresed and analyzed by WB using antibodies against TLR4 (BA1717, Boster, Wuhan, China), MYD88 (abs135682, Absin, Shanghai, China), IKBα (D120138, Sangon Biotech, Shanghai, China), p-IKBα (D151548, Sangon Biotech, Shanghai, China), p-p65 (HZ4902812, TW reagent, Shanghai, China), TJP-1 (mAb13663, Cell Signaling, USA), Occludin (ab167161, Abcam, USA), and actin (60008, Proteintech, USA).

Techniques: Quantitative Proteomics, Staining

Journal: Cell reports. Medicine

Article Title: A CD36-dependent non-canonical lipid metabolism program promotes immune escape and resistance to hypomethylating agent therapy in AML.

doi: 10.1016/j.xcrm.2024.101592

Figure Lengend Snippet: Figure 3. OxLDL and PA synergize to suppress T cell activity via CD36-mediated innate immune signaling in AML cells (A and B) THP-1 cells (A) or MV4-11 cells (B) stimulated in CM or LDM supplied with different lipids (palmitate [PA], 20 mM; OxLDL, 25 mg/mL) for 3 days were further used for T cell proliferation assay (n = 3). (C–E) Immunoblot analysis of TLR4 (C and E) or LYN (D) precipitated proteins. (C and E) AML cells were treated with different reagents (PA, 20 mM; OxLDL, 25 mg/mL; PP1, 10 mM) for 5 h before immunoprecipitation. (F) MV4-11 cells treated with LDM supplied with different reagents (OxLDL, 25 mg/mL; TAK-242, 10 mM; PP1, 10 mM) for 3 days were further used for T cell proliferation assay (n = 3). (G–I) AML cells labeled with a chemical probe (alk-C16) for 4 h in the absence (G and H) or presence of SSO (50 mM) (I) were used for palmitoylation assay.

Article Snippet: After blocking with 5% nonfat milk in TBST, the membranes were incubated with the the following antibodies overnight 4 C with constant rotation: p-p65 (Cell Signaling Technology; 3033S), p65 (Cell Signaling Technology; 8242S), actin (Cell Signaling Technology; 3700S), Lamin-B1 (Santa Cruz Biotechnology; sc-374015), MYD88 (Cell Signaling Technology; 4283), CD36 (abcom; ab133625), ZDHHC6 (abcom; ab121423), TLR4 (CUSABIO Technology; CSB-PA001434), LYN (Cell Signaling Technology; 2796S).

Techniques: Activity Assay, Proliferation Assay, Western Blot, Immunoprecipitation, Labeling

Journal: Frontiers in Immunology

Article Title: CEACAM 1, 3, 5 and 6 -positive classical monocytes correlate with interstitial lung disease in early systemic sclerosis

doi: 10.3389/fimmu.2022.1016914

Figure Lengend Snippet: Antibodies used for Mass Cytometry.

Article Snippet: TLR4 , HTA125 , 158Gd , Fluidigm.

Techniques: Cytometry

Journal: FEMS Immunology & Medical Microbiology

Article Title: Toll-like receptor 4 signaling plays a role in triggering periodontal infection

doi: 10.1111/j.1574-695x.2008.00386.x

Figure Lengend Snippet: Fig. 1. Expression of TLR4 mRNA in HPDLCs. Four passage HPDLCs were obtained as described in the ‘Materials and methods’. Total RNA was extracted, and the mRNA expression of TLR4 was analyzed by reverse transcriptase PCR (a). Levels of b-actin mRNA served as internal controls (b). Lanes: M, molecular size marker; 1, sample A; 2, sample B; 3, sample C; 4, negative controls. The results were representative of three different experiments.

Article Snippet: The endogenous peroxidase signal was quenched by incubation in 1.5% H2O2 for 15 min. After 30 min of blocking with normal goat serum, the cells were incubated with mouse anti-TLR4 mAb (Serotec) at a dilution of 1 : 200 overnight at 4 1C.

Techniques: Expressing, Reverse Transcription, Marker

Journal: FEMS Immunology & Medical Microbiology

Article Title: Toll-like receptor 4 signaling plays a role in triggering periodontal infection

doi: 10.1111/j.1574-695x.2008.00386.x

Figure Lengend Snippet: Fig. 2. Expression of TLR4 in HPDLCs detected by immunofluorescence staining. Four passage HPDLCs from three individual healthy subjects were obtained as described in the ‘Materials and methods’. After fixation, the cells were treated with monoclonal antibody to TLR4 (anti- TLR4 mAb) (green), and the nuclei were counterstained with PI (red) (a). The other cells were treated with PBS, which was substituted for anti- TLR4 mAb, and served as controls (b). Scale bars = 20 mm.

Article Snippet: The endogenous peroxidase signal was quenched by incubation in 1.5% H2O2 for 15 min. After 30 min of blocking with normal goat serum, the cells were incubated with mouse anti-TLR4 mAb (Serotec) at a dilution of 1 : 200 overnight at 4 1C.

Techniques: Expressing, Staining

Journal: FEMS Immunology & Medical Microbiology

Article Title: Toll-like receptor 4 signaling plays a role in triggering periodontal infection

doi: 10.1111/j.1574-695x.2008.00386.x

Figure Lengend Snippet: Fig. 3. Gene expression changes in HPDLCs stimulated with lipopolysaccharide analyzed by real-time PCR. HPDLCs were cultured in DMEM with or without 1 mg mL1 lipopolysaccharide for 6 h. cDNA was prepared. Real-time PCR was used to quantify TLR4 (a), IL-6 (b), Fos (c) and LY64 (d) mRNA expression levels. For each transcript, conventional PCR-amplified products were used as standards (from 1 100 to 1 106) to generate a standard curve for absolute real-time quantification. The absolute mRNA levels of all the genes were then normalized to b-actin levels of individual samples. Three different experiments were performed, and the representative results were shown. HPDLCs from three individual persons were pooled for each experiment. Lipopolysaccharide induced the expression of TLR4 and IL-6, while it repressed the expression of Fos and LY64.

Article Snippet: The endogenous peroxidase signal was quenched by incubation in 1.5% H2O2 for 15 min. After 30 min of blocking with normal goat serum, the cells were incubated with mouse anti-TLR4 mAb (Serotec) at a dilution of 1 : 200 overnight at 4 1C.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Cell Culture, Expressing

Journal: FEMS Immunology & Medical Microbiology

Article Title: Toll-like receptor 4 signaling plays a role in triggering periodontal infection

doi: 10.1111/j.1574-695x.2008.00386.x

Figure Lengend Snippet: Fig. 4. Secretion of IL-6 in HPDLCs stimulated with lipopolysaccharide in the absence or presence of anti-TLR4 mAb. (a) HPDLCs were plated at a density of 5 104 cells mL1 in 96-well plates. After 2 days, the cells were exposed to Escherichia coli lipopolysaccharide (100 ng mL1, 1 mg mL1, 10 mg mL1). Following incubation for various periods of time (6, 12, 24, 48 h) at 37 1C, the supernatants were harvested and assayed via ELISA for the concentration of IL-6. (b) HPDLCs (5 104 cells mL1 in 96-well plates) were incubated with 1 mg mL1 lipopolysaccharide for 24 h in the absence or presence of various titers of anti-TLR4 mAb (500–25-fold dilutions). The levels of IL-6 in the culture supernatants were measured by ELISA. P o 0.05, significantly different from levels in the absence of anti-TLR4 mAb. Three different experiments were performed, and each data point represents the mean SD. HPDLCs from three individual people were pooled for each experiment.

Article Snippet: The endogenous peroxidase signal was quenched by incubation in 1.5% H2O2 for 15 min. After 30 min of blocking with normal goat serum, the cells were incubated with mouse anti-TLR4 mAb (Serotec) at a dilution of 1 : 200 overnight at 4 1C.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay