Journal: Neural Regeneration Research

Article Title: Role of Toll-like receptor 4 in inflammatory reactions of hippocampal neurons ★

doi: 10.3969/j.issn.1673-5374.2013.16.003

Figure Lengend Snippet: Effect of lipopolysaccharide (LPS) on the expression of nuclear factor-kappa B (NF-κB) p65 and phospho-inhibitor of kappa B α (p-IκBα) protein at 0, 0.5, 2, 6 and 12 hours after LPS (10 μg/mL) stimulation determined by western blot analysis. (A) NF-κB p65 protein level in the nuclei of hippocampal neurons was detected by western blot analysis. Histone was used as an internal control in the nuclei. (B) p-IκBα protein level in the cytosol of each group was detected by western blot analysis. β-actin was used as an internal control. (C) Quantification of NF-κB p65 and p-IκBα protein expression. The mean densitometry values of NF-κB p65/histone and p-IκBα/β-actin in various groups were analyzed and expressed as bar charts to represent the relative expression of NF-κB p65 protein in the nuclei and p-IκBα protein in cytosol. a P < 0.01, vs . control group (0 h). Data are expressed as mean ± SD and n = 3 per group. One-way analysis of variance and comparisons between two groups were made with the least significant difference and Student-Newman-Keuls post-hoc test. h: Hour.

Article Snippet: Each membrane was incubated in fresh blocking buffer (containing 5% nonfat dry milk) at room temperature for 30 minutes, and was then incubated with rabbit anti-Toll-like receptor 4 polyclonal antibody (1:200; Santa Cruz Biotechnology), mouse anti-β-actin monoclonal antibody (1:2 000; Santa Cruz Biotechnology), mouse anti-histone H1 monoclonal antibody (1:500; Millipore, Billerica, MA, USA), rabbit anti-nuclear factor-kappa B p65 polyclonal antibody (1:1 000; Kangchen BIO-TECH, Shanghai, China) and mouse anti-phospho-inhibitor of kappa B α monoclonal antibody (1:1 000, Kangchen BIO-TECH) at 4°C overnight.

Techniques: Expressing, Western Blot

Journal: Neural Regeneration Research

Article Title: Role of Toll-like receptor 4 in inflammatory reactions of hippocampal neurons ★

doi: 10.3969/j.issn.1673-5374.2013.16.003

Figure Lengend Snippet: Effect of Toll-like receptor 4 (TLR4) antibody on the expression of nuclear factor-kappa B (NF-κB) p65, phospho-inhibitor of kappa B α (p-IκBα) and the production of interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). (A) Cells were pretreated with TLR4 antibody (10 μg/mL) for 1 hour and were then stimulated with lipopolysaccharide (LPS; 10 μg/mL) for 2 hours. NF-κB p65 protein (65 kDa) levels in the nucleus of hippocampal neurons of different groups were detected by western blot analysis. Histone (33 kDa) was used as an internal control in the nuclei. (B) p-IκBα protein (41 kDa) level in the cytosol of each group was detected by western blot analysis. β-actin (43 kDa) was used as an internal control. (C) Relative levels of NF-κB p65 in the nucleus and p-IκBα in the cytosol. The mean densitometry values of NF-κB p65/histone and p-IκBα/β-actin in various groups were analyzed and expressed as bar charts to represent the relative expression of NF-κB p65 protein in the nucleus and p-IκBα protein in the cytosol. a P < 0.01, vs . control group; b P < 0.05, vs . LPS group. (D) Effect of TLR4 antibody on the concentration of IL-1β and TNF-α in culture supernatant. Cells were pretreated with TLR4 antibody (10 μg/mL) for 1 hour and were then stimulated with LPS (10 μg/mL) for 24 hours. The concentration of IL-1β and TNF-α in culture supernatant was detected by enzyme-linked immunosorbent assay. a P < 0.01, vs . control group; b P < 0.01, vs . LPS group. (C–D) Data are expressed as mean ± SD and n = 3 per group. One-way analysis of variance and comparisons between two groups were made with the least significant difference and Student-Newman-Keuls post-hoc test.

Article Snippet: Each membrane was incubated in fresh blocking buffer (containing 5% nonfat dry milk) at room temperature for 30 minutes, and was then incubated with rabbit anti-Toll-like receptor 4 polyclonal antibody (1:200; Santa Cruz Biotechnology), mouse anti-β-actin monoclonal antibody (1:2 000; Santa Cruz Biotechnology), mouse anti-histone H1 monoclonal antibody (1:500; Millipore, Billerica, MA, USA), rabbit anti-nuclear factor-kappa B p65 polyclonal antibody (1:1 000; Kangchen BIO-TECH, Shanghai, China) and mouse anti-phospho-inhibitor of kappa B α monoclonal antibody (1:1 000, Kangchen BIO-TECH) at 4°C overnight.

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Quercetin Attenuates Trauma-Induced Heterotopic Ossification by Tuning Immune Cell Infiltration and Related Inflammatory Insult

doi: 10.3389/fimmu.2021.649285

Figure Lengend Snippet: SIRT1/NFκB signaling was responsible for the dysregulated immune responses in trauma-induced HO. (A) Schematic depiction of the agonist treatment protocols for animal experiments. (B, D) Observation and quantification of HO formation using micro-CT. (C) Fluorescence staining of SIRT1 7 days after burn/tenotomy. (E) Toluidine blue staining for observation of mast cells 7 days after burn/tenotomy. (F) Double fluorescence staining of CD11b and F4/80 7 days after burn/tenotomy. (G) Double fluorescence staining of tryptase and acetyl-p65 7 days after burn/tenotomy. (H) Double fluorescence staining of F4/80 and acetyl-p65 7 days after burn/tenotomy. (I) Quantification of the mast cells in toluidine blue staining images. (J) Quantification of CD11b+F4/80+ cells in double fluorescence staining images. (K) Relative gene expression of the mast cell marker CPA3 as determined by qRT-PCR. (L) Relative gene expression of macrophage marker F4/80 as determined by qRT-PCR. Original magnification is 40x. Inserts are approximately 3.5x magnified images of the boxed area. N=4/group. ***P < 0.001.

Article Snippet: Anti-NFkB p65 (acetyl Lys310) antibody (Cat# GTX86963) was purchased from Genetex (CA, USA).

Techniques: Micro-CT, Fluorescence, Staining, Gene Expression, Marker, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Quercetin Attenuates Trauma-Induced Heterotopic Ossification by Tuning Immune Cell Infiltration and Related Inflammatory Insult

doi: 10.3389/fimmu.2021.649285

Figure Lengend Snippet: Quercetin targets and modulates SIRT1 to reduce NFκB acetylation during immune response modulation. (A) Ramachandran plot for homology-modeled mouse SIRT1. The core area was colored red and involved 94.2% of the amino acid residues, while the allowed area was colored yellow and involved 5.8% of the amino acid residues. None of the amino acid residues were located in the generous (light yellow) or disallowed (white) areas. (B) Tables listing the nine top binding affinities of quercetin or SRT1720 to SIRT1. Affinity denotes binding energy, RMSD represents root-mean-square deviation and RMSE represents root-mean-square error. (C) Binding and interaction sites of quercetin with SIRT1 are shown. Cells were pretreated with 5 μM, 10 μM, or 20 μM quercetin for 1 h, and then incubated with respective stimulus for 1 h or 24 h. The effects of quercetin on SIRT1 and acetylated p65 expression in (D) THP-1, (E) BMDMs and (F) P815 cells during their activation were assessed by western blot. The relative level of SIRT1 and acetyl-p65 in (G) THP-1, (H) BMDMs and (I) P815 cells quantified using ImageJ and normalized to β-actin are shown. Cells were pretreated with 20 μM quercetin for 1 h, 10 μM EX527 and 1 μM SRT1720 HCI for 6 h and then incubated with respective stimulus for 1 h or 24 h. The effects of SIRT1 activation and inhibition on acetylated p65 expression in (J) THP-1, (K) BMDMs and (L) P815 cells during their activation were assessed by western blot. The relative level of acetylated p65 in (M) THP-1, (N) BMDMs and (O) P815 cells quantified using ImageJ and normalized to β-actin is shown. (P, Q) Immunofluorescence staining showing the expression of SIRT1 and acetylated p65 in the tissues 7 days after tenotomy. Original magnification is 40x. Inserts are approximately 3.5x magnified images of the boxed area. N=4/group. *P < 0.05.

Article Snippet: Anti-NFkB p65 (acetyl Lys310) antibody (Cat# GTX86963) was purchased from Genetex (CA, USA).

Techniques: Binding Assay, Incubation, Expressing, Activation Assay, Western Blot, Inhibition, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Quercetin Attenuates Trauma-Induced Heterotopic Ossification by Tuning Immune Cell Infiltration and Related Inflammatory Insult

doi: 10.3389/fimmu.2021.649285

Figure Lengend Snippet: Quercetin impeded immune cell infiltration and HO by activating SIRT1 in vivo . (A) Schematic depiction of the indicated treatment protocols for animal experiments. (B) Micro-CT scanning for observation and quantification of HO formation. (C) Double fluorescence staining of CD11b and F4/80 7 days after burn/tenotomy and quantification of CD11b+F4/80+ cells. (D) Toluidine blue staining for observation and quantification of mast cells 7 days after burn/tenotomy. (E) Relative gene expression of the mast cell marker CPA3 as determined by qRT-PCR. (F) Double fluorescence staining of tryptase and acetyl-p65 7 days after burn/tenotomy. (G) Relative gene expression of macrophage marker F4/80 as determined by qRT-PCR. (H) Double fluorescence staining of F4/80 and acetyl-p65 7 days after burn/tenotomy. Original magnification is 40x. Inserts are approximately 3.5x magnified images of the boxed area. N=4/group. ***P < 0.001.

Article Snippet: Anti-NFkB p65 (acetyl Lys310) antibody (Cat# GTX86963) was purchased from Genetex (CA, USA).

Techniques: In Vivo, Micro-CT, Fluorescence, Staining, Gene Expression, Marker, Quantitative RT-PCR