Journal: Frontiers in Immunology

Article Title: MicroRNA-1 Negatively Regulates Peripheral NK Cell Function via Tumor Necrosis Factor-Like Weak Inducer of Apoptosis (TWEAK) Signaling Pathways During PPRV Infection

doi: 10.3389/fimmu.2019.03066

Figure Lengend Snippet: Regulation of NK cell function by miR-1 is mediated by TWEAK signaling. (A) Goat CD16 + CD14 − NK cells were transfected with control miRNA (MC) or miR-1 mimics, or miR-1 mimics and incubated with recombinant TWEAK at the final concentration of 50 nM. Forty-eight hours later, the cells were infected with PPRV at an MOI of 1, and 24 h later, the protein expression of NKG2D, perforin, IFN-γ, and TNF-α was measured by Western blot. Histograms of the right panel showed densitometric analysis of indicated protein normalized to GAPDH to correct for protein loading. (B) Goat CD16 + CD14 − NK cells were transfected with lentivirus TWEAK, si-TWEAK, or respective negative control. Forty-eight hours later, the cells were infected with PPRV at an MOI of 1, and 24 h later, the protein levels of SOCS-1, MyD88, STAT3, pSTAT3, and NF-κB p65 in NK cells were determined by Western blot and normalized to the levels of GAPDH. Results are expressed as means ± standard error of mean (SEM). P -values were calculated using Student's t -test. An asterisk indicates a comparison with the indicated control. * P < 0.05; ** P < 0.01. n.s., not significant.

Article Snippet: For downstream signaling pathway detection, membranes were probed overnight with rabbit anti-SOCS1 (Sangon Biotech, Shanghai, China), rabbit anti-NF-κB p65 (Bioss, Beijing, China), rabbit anti-MyD88 (Bioss, Beijing, China), rabbit anti-STAT3 (Bioss, Beijing, China), or rabbit anti-pSTAT3 (Bioss, Beijing, China).

Techniques: Cell Function Assay, Transfection, Incubation, Recombinant, Concentration Assay, Infection, Expressing, Western Blot, Negative Control

Journal: International journal of immunopathology and pharmacology

Article Title: Corilagin relieves atherosclerosis via the toll-like receptor 4 signaling pathway in vascular smooth muscle cells.

doi: 10.1177/03946320241254083

Figure Lengend Snippet: Figure 2. Effect of ox-LDL on the TLR4 signaling pathway in MOVAS cells. (a) mRNA expression of TLR4, TIRAP and MyD88 was measured by qRT-PCR. *p < .05 compared with other concentrations of ox-LDL groups or control group as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (b) *p < .05 compared with either time point or control groups determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5).

Article Snippet: Afterwards, the cells were subjected to a 2-h incubation at 37°C with either primary anti-TLR4 antibody (1:200 dilution; Cat No. BA1717; Boster Biological Technology, Pleasanton, CA, USA) or anti-MyD88 antibody (1:400 dilution; Cat No. PB9148; Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: International journal of immunopathology and pharmacology

Article Title: Corilagin relieves atherosclerosis via the toll-like receptor 4 signaling pathway in vascular smooth muscle cells.

doi: 10.1177/03946320241254083

Figure Lengend Snippet: Figure 3. Effect of corilagin on the TLR4 signaling pathway in MOVAS cells stimulated by ox-LDL. (a) mRNA expression of TLR4, TIRAP, MyD88, TRAF6, p38, NEMO and IRF5 was measured by qRT-PCR. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group, **p < .05 compared with the aspirin group as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (B. C) Protein abundance was measured by western blotting. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group, **p < .05 compared with the aspirin group, as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (D, E) Abundance of IL-6 and MCP-1 in cell culture supernatant was measured by ELISAs. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group, **p < .05 compared with the aspirin group as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (F) Effect of corilagin on the MOVAS cells proliferation. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (G, H) apoptosis ratio of MOVAS were detected by Annexin V staining. No significant difference was found among four groups determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5).

Article Snippet: Afterwards, the cells were subjected to a 2-h incubation at 37°C with either primary anti-TLR4 antibody (1:200 dilution; Cat No. BA1717; Boster Biological Technology, Pleasanton, CA, USA) or anti-MyD88 antibody (1:400 dilution; Cat No. PB9148; Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Control, Quantitative Proteomics, Western Blot, Cell Culture, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Change in Oxidative Stress and Mitochondrial Dynamics in Response to Elevated Cold-Inducible RNA-Binding Protein in Cardiac Surgery-Associated Acute Kidney Injury

doi: 10.1155/2022/3576892

Figure Lengend Snippet: CPB induced mitochondrial dynamics disorder and increased inflammatory factor secretion. (a–f) Expression of IL-6, IL-1 β , and TNF- α in the serum at 0 and 4 hours after CPB. Compared to the CPB group, C23 administration decreased the expression of IL-6, IL-1 β , and TNF- α by 54.0%, 67.5%, and 45.2% at 4 hours, respectively. (g) Western blot analysis of renal Bax, Bcl-2, and cleaved-caspase-3 expression. (h) CPB upregulated the NADPH oxidase via the TLR-4/MyD88 pathway, which promoted the expressions of mitochondrial fission-related proteins Fis1 and Drp1 and reduced the expression of fusion-related protein Mfn2. ∗ P < 0.05 vs. sham; # P < 0.05 vs. CPB.

Article Snippet: The other antibodies used in this study included TLR-4 (19811-1-AP, Proteintech, 1 : 1000), MyD88 (BA2321, Boster, 1 : 500), and anti- β -actin (4967, Cell Signaling Technology, 1 : 1000).

Techniques: Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Change in Oxidative Stress and Mitochondrial Dynamics in Response to Elevated Cold-Inducible RNA-Binding Protein in Cardiac Surgery-Associated Acute Kidney Injury

doi: 10.1155/2022/3576892

Figure Lengend Snippet: Putative mechanism of CIRP in cardiac surgery-associated acute kidney injury. During cardiac surgery, CIRP is secreted into the circulation in response to hypothermia and hemodynamics change. Extracellular CIRP promotes the expression of NADPH oxidase in renal tubular epithelial cells via the TLR-4/MyD88 pathway and aggravates intracellular oxidative stress. ROS accumulation induces mitochondrial dynamics disorder, which ultimately increases apoptosis and promotes AKI. CIR: cold-inducible RNA-binding protein; NADPH: nicotinamide adenine dinucleotide phosphate; ROS: reactive oxygen species.

Article Snippet: The other antibodies used in this study included TLR-4 (19811-1-AP, Proteintech, 1 : 1000), MyD88 (BA2321, Boster, 1 : 500), and anti- β -actin (4967, Cell Signaling Technology, 1 : 1000).

Techniques: Expressing, RNA Binding Assay

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Re inhibits myocardial fibrosis by regulating miR-489/myd88/NF-κB pathway

doi: 10.1016/j.jgr.2021.11.009

Figure Lengend Snippet: MiR-489 regulated myd88/NF-κB pathway in CFs. (A, B) The expression of myd88, NF-κB p65 and p-p65 were analyzed in CFs transfected with miR-489 mimic, ∗ P < 0.01 vs Normal group; # P ＜0.01 vs mimic group. (C, D) The expression of myd88, NF-κB p65 and p-p65 were analyzed in CFs transfected with miR-489 inhibitor, ∗ P < 0.01 vs Normal group; § P < 0.05 vs Normal group; # P ＜0.01 vs inhibitor group; & P ＜0.05 vs inhibitor group. All data are presented as the mean ± SD (n = 3).

Article Snippet: Rabbit anti-myd88 (1:1000; Abbkine), rabbit anti–NF–κB p65(1:1000; SAB), rabbit anti-p–NF–κB p65 (1:1000; Immunoway), anti-collagenI (1:1000; Proteintech), rabbit anti-collagen III (1:1000; Proteintech), rabbit anti-α-SMA (1:1000; CST), rabbit anti-GAPDH (1:5000; Abbkine).

Techniques: Expressing, Transfection

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Re inhibits myocardial fibrosis by regulating miR-489/myd88/NF-κB pathway

doi: 10.1016/j.jgr.2021.11.009

Figure Lengend Snippet: G-Re regulates the miR-489/myd88/NF-κB pathway. (A, B) The expression miR-489 was measured in CFs treated by AngⅡ and the myocardium of AMI mice. (C, D) The expression of myd88 was analyzed in the myocardium of AMI mice and CFs treated by AngⅡ. (E, F) The expression of NF-κB p65 and p-p65 were analyzed in the myocardium of AMI mice and CFs treated by AngⅡ, ∗ P < 0.01 vs Normal group, # P ＜0.01 vs Model group. All data are presented as the mean ± SD (n = 3).

Article Snippet: Rabbit anti-myd88 (1:1000; Abbkine), rabbit anti–NF–κB p65(1:1000; SAB), rabbit anti-p–NF–κB p65 (1:1000; Immunoway), anti-collagenI (1:1000; Proteintech), rabbit anti-collagen III (1:1000; Proteintech), rabbit anti-α-SMA (1:1000; CST), rabbit anti-GAPDH (1:5000; Abbkine).

Techniques: Expressing

Journal: CNS Neuroscience & Therapeutics

Article Title: Intracerebral hemorrhage‐induced brain injury in mice: The role of peroxiredoxin 2‐Toll ‐like receptor 4 inflammatory axis

doi: 10.1111/cns.14681

Figure Lengend Snippet: TLR (toll‐like receptor) signaling pathway is upregulated in the inflammation induced by Prx2 (peroxiredoxin 2). (A) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of DEGs (differentially expressed genes) in the ipsilateral basal ganglia at 24 h after saline or Prx2 injection (10 μL); n = 5 for saline and n = 7 for Prx2. Dot color represents Q‐value from the least significant (green) to the most significant (red); dot size represents gene number, the number of significant DEGs in the KEGG pathway; rich factor represents the fraction of significant DEGs among all genes in the KEGG pathway. (B) FPKM (Mean fragments per kilobase of transcript per million mapped reads) values of TLR2, TLR4, Myd88 (myeloid differentiation primary response protein 88) in the ipsilateral basal ganglia at 24 h after saline or Prx2 injection. Values are mean ± SD; n = 5 for saline and n = 7 for Prx2; # p < 0.01 versus saline group. (C) TLR4 immunofluorescence at the ipsilateral basal ganglia 24 h after saline or Prx2 injection. Values are mean ± SD; n = 7 for both groups; # p < 0.01 versus saline group. Scale bar = 200 μm at low magnification, 20 μm at high magnification.

Article Snippet: The primary antibodies used for immunohistochemical staining and immunofluorescence staining were rabbit anti‐Prx2 IgG (10545‐2‐AP, 1:200; Proteintech, Wuhan, China), rabbit anti‐IBA‐1 (ionized calcium‐binding adaptor molecule 1, 10904‐1‐AP, 1:400; Proteintech), rabbit anti‐MPO IgG (myeloperoxidase, PA5‐16672, 1:400; ThermoFisher Scientific), and goat anti‐MyD88 (myeloid differentiation primary response protein 88, EB06667, 1:200; Everest Biotech, Oxford, UK).

Techniques: Saline, Injection, Immunofluorescence

Journal: CNS Neuroscience & Therapeutics

Article Title: Intracerebral hemorrhage‐induced brain injury in mice: The role of peroxiredoxin 2‐Toll ‐like receptor 4 inflammatory axis

doi: 10.1111/cns.14681

Figure Lengend Snippet: Intracaudate injection of Prx2 (peroxiredoxin 2) activates TLR4 (toll‐like receptor 4) signaling pathway. (A) Triple immunofluorescence labeling of IBA‐1 (ionized calcium‐binding adaptor molecule 1) and TLR4 at the ipsilateral basal ganglia 24 h after Prx2 injection. Co‐localization of TLR4 with IBA‐1 (microglia/macrophage marker). Scale bar = 200 μm at low magnification, 20 μm at high magnification, 10 μm at the highest magnification. (B) Triple immunofluorescence labeling of MPO (myeloperoxidase) and TLR4 at the ipsilateral basal ganglia 24 h after Prx2 injection. Co‐localization of TLR4 with MPO (neutrophil marker). Scale bar = 200 μm at low magnification, 20 μm at high magnification, 5 μm at the highest magnification. (C) Triple immunofluorescence labeling of TLR4 and Myd88 (myeloid differentiation primary response protein 88) at the ipsilateral basal ganglia 24 h after saline or Prx2 injection. Values are mean ± SD; n = 7 for both groups; # p < 0.01 versus saline group. Scale bar = 200 μm at low magnification, 20 μm at high magnification.

Article Snippet: The primary antibodies used for immunohistochemical staining and immunofluorescence staining were rabbit anti‐Prx2 IgG (10545‐2‐AP, 1:200; Proteintech, Wuhan, China), rabbit anti‐IBA‐1 (ionized calcium‐binding adaptor molecule 1, 10904‐1‐AP, 1:400; Proteintech), rabbit anti‐MPO IgG (myeloperoxidase, PA5‐16672, 1:400; ThermoFisher Scientific), and goat anti‐MyD88 (myeloid differentiation primary response protein 88, EB06667, 1:200; Everest Biotech, Oxford, UK).

Techniques: Injection, Immunofluorescence, Labeling, Binding Assay, Marker, Saline