Journal: Oncology Letters

Article Title: SLAMF6/Ly108 promotes the development of hepatocellular carcinoma via facilitating macrophage M2 polarization

doi: 10.3892/ol.2022.13203

Figure Lengend Snippet: Ly108 promotes hepatocellular carcinoma growth by inhibiting the NF-κB signaling pathway. (A) A murine HCC model was prepared (n=6) and the resulting tumors were weighed at the time of sacrifice. Tumor images are presented in the left panel, while summary data are presented in the right panel. Tumor growth was measured via tumor size, as presented in the middle panel. (B) PMs were transfected with negative control siRNA or Ly108 siRNA, with or without Bay11-7082 prior to treatment with 20 ng/ml IL-4. Western blotting was performed to determine p65 and p-p65 expression. (C) Ly108 siRNA transfection and Bay11-7082 or control DMSO stimulation prior to IL-4 induction was performed on murine PMs. Reverse transcription quantitative PCR was performed to determine the relative expression of arg-1, TNF-α, IL-1β and iNOS. (D) The macrophage culture supernatants in were collected and co-cultured with Hepa1–6 cells for the transwell assays. Clonogenic formation assays were then performed. *P<0.05, **P<0.01 and ***P<0.001. HCC, hepatocellular carcinoma; NC, negative control; si, small interfering; Arg-1, arginase-1; IL-1β, interleukin 1β; iNOS, inducible nitric oxide synthase; TNF-α, tumor necrosis factor-α; NF-κB, nuclear factor-κB; IL-4, interleukin 4; Ctr, control; p, phosphorylated; PMs, peritoneal macrophages.

Article Snippet: After blocking membranes with 5% bovine serum albumin (cat. no. A8010; Beijing Solarbio Science & Technology Co., Ltd.) for 2 h at room temperature, membranes were incubated with primary antibodies against phosphorylated (p)-NF-κB p65 (Cell Signaling Technology, Inc.; cat. no. 3033; 1:1,000); NF-κB (Cell Signaling Technology, Inc.; cat. no. 8242; 1:1,000) and β-actin (Cell Signaling Technology, Inc.; cat. no. 3700; 1:1,000) at 4°C overnight.

Techniques: Transfection, Negative Control, Western Blot, Expressing, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Culture

Journal: Glia

Article Title: Lipid transporter Spns2 promotes microglia pro-inflammatory activation in response to amyloid-beta peptide

doi: 10.1002/glia.23558

Figure Lengend Snippet: Spns2 enhanced microglia pro-inflammatory polarization partly through NFκB signaling. A. Spns2KO reduced Aβ42-induced nuclear pP65. *, p<0.05. N=3. The bands were quantified by densitometry using ImageJ and normalized to Histone H3 (HisH3). B. Spns2KO significantly reduced Aβ42-induced pIκB level. The bands were quantified using ImageJ and normalized to GAPDH. **, p<0.01. N=3. C and D, S1P coordinated with Aβ42 to induce NFκB activity determined by Western blot (C) and densitometry analysis (D). *, p<0.05. N=3.

Article Snippet: Materials: The antibody against phosphorylated NFκB P65 (pP65) (Ser 536) and LPS were from Sigma Aldrich (St. Louis, MO), antibodies against GAPDH, pIκB, TNFα, IL4, IL6, and IL10 were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Activity Assay, Western Blot

Journal: JCI Insight

Article Title: Remote ischemic preconditioning causes transient cell cycle arrest and renal protection by a NF- κ B–dependent Sema5B pathway

doi: 10.1172/jci.insight.158523

Figure Lengend Snippet: ( A ) Isolated murine renal tubular epithelial cells were incubated with HMBG1 (0.1 μg/mL) or HMGB1 plus TLR4 inhibitor (TAK-242). Activation of NF-κB and AMPKα was detected by Western blotting for NF-κB p-p65 and β-actin as loading control, as well as p-AMPKα and total AMPKα (exemplary blots). Isolated murine renal tubular epithelial cells were incubated with HMBG1 (0.1 μg/mL) or HMGB1 plus NF-κB inhibitor (Bay-117082). ( B and C ) TIMP-2 and IGFBP7 were analyzed by ELISAs ( n = 6). ( D ) Isolated murine renal tubular epithelial cells were treated with HMBG1 (0.1 μg/mL) or HMGB1 in combination with NF-κB inhibitor (Bay-117082). The proportion of cells in G 0 /G 1 phase was analyzed by measuring cellular DNA content by flow cytometry ( n = 6). After induction of general anesthesia WT mice received either 3 cycles RIPC or control procedure. Some mice received a NF-κB inhibitor (Bay-117082, 10 mg/kg i.p.) or AMPKα inhibitor before RIPC. Twenty-four hours after IRI induction, mice were sacrificed. ( E ) The recruitment of neutrophils (PMNs) into the kidney was analyzed by flow cytometry ( n = 4). ( F ) Serum creatinine levels were measured by a photometric assay ( n = 4). ( G ) The biomarkers TIMP-2 and IGFBP7 were measured in urine samples 24 hours after inducing renal IRI. ( H ) Renal tubular injury score was assessed based on histology ( n = 4). One-way ANOVA followed by Bonferroni testing was used for statistical analysis; * P < 0.05.

Article Snippet: Lysates were boiled with Laemmli sample buffer, run on 10% SDS-PAGE gels and immunoblotted using antibodies against NF-κB p-p65 (clone 93H1, Cell Signaling Technology), pAMPKα (clone D4D6D, Cell Signaling Technology), AMPKα (clone D63G4, Cell Signaling Technology), and p38 (catalog 9212, Cell Signaling Technology); Semaphorin 5b (catalog NBP2-56604, Novus Biologicals); or β-actin (clone AC-15, Sigma-Aldrich).

Techniques: Isolation, Incubation, Activation Assay, Western Blot, Control, Flow Cytometry

Journal: Mediators of Inflammation

Article Title: Interference in Macrophage Balance (M1/M2): The Mechanism of Action Responsible for the Anti-Inflammatory Effect of a Fluorophenyl-Substituted Imidazole

doi: 10.1155/2024/9528976

Figure Lengend Snippet: Effect of fluorophenyl-imidazole on TLR-4 receptor (CD284-MD2) (a); mannose receptor (CD206 hi+ ) (b); phosphorylated protein p65 (c); apoptosis (d); and phagocytic activity (e) in RAW 264.7 macrophages stimulated with LPS (1 μ g/mL). The experimental groups used were B: cells pretreated with vehicle and incubated with PBS (blank group); LPS: cells pretreated with vehicle and stimulated with LPS (1 μ g/mL); dexa + LPS: cells pretreated with dexamethasone (7 μ M) and stimulated with LPS (1 μ g/mL); flu (1 μ M): cells pretreated with the best concentration (based on IC 50 values) (1 µ M) of fluorophenyl-imidazole alone; and (flu 1 μ M) + LPS: fluorophenyl-imidazole (1 µ M), before LPS (1 μ g/mL). Taxel (30 μ M) was used alone in apoptosis experiments, as positive control of apoptosis. The results for TLR-4 (CD284-MD2) and (CD206 hi+ ) were quantified as receptor expression percentage in relation to cells stained with control isotype antibody while, phosphorylated protein p65 expression was compared to the blank control (cells with no treatment) that represents the basal expression phosphorylated p65 protein. The apoptosis values (%) were quantified as the percentage of apoptotic cells compared with the total cell number and the phagocytic index was measured using the following equation: phagocytic index (PI) = A 1/ A 0; where A 1 is the absorbance of sample, LPS and dexamethasone, and A 0 is the absorbance of black control. The results were expressed as the mean ± SEM; n = 3; ns, not significant; ∗∗ P < 0.01; and ∗∗∗ P < 0.001.

Article Snippet: Afterward, they were transferred to ELISA microplates containing monoclonal antibodies specific against the phosphorylated p 65 protein (PathScan®Phospho-NF- κ B p65 (Ser536) ELISA Kit (Cell Signalling Technology, Inc., Danvers, Massachusetts, USA)).

Techniques: Activity Assay, Incubation, Concentration Assay, Positive Control, Expressing, Staining, Control

Journal: Poultry Science

Article Title: Glycerol monolaurate regulates apoptosis and inflammation by suppressing lipopolysaccharide-induced ROS production and NF-κB activation in avian macrophages

doi: 10.1016/j.psj.2024.103870

Figure Lengend Snippet: GML inhibited the activation of the NF-κB signaling pathway in LPS-treated HD11 cells (n = 4). RT-qPCR analysis of the relative mRNA expression of (A) TLR4 and (B) NF-κB p65 in HD11 cells after 12 h incubation with GML (10 μg/mL) followed by 12 h LPS (2 μg/mL) treatment. (C-F) Immunoblot analysis of phosphorylated NF-κB p65, NF-κB p65, and GAPDH (loading control) in HD11 cells after 12 h incubation with GML (10 μg/mL) followed by 12 h LPS (2 μg/mL) treatment. * P < 0.05, ⁎⁎ P < 0.01, and ⁎⁎⁎ P < 0.001. One-way ANOVA with Tukey's multiple comparisons. Abbreviations: GML, glycerol monolaurate; LPS, lipopolysaccharide, TLR4, toll-like reporter 4; NF-κB, nuclear factor kappa-B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The membranes were blocked, followed by incubation with primary antibodies against p-NF-κB p65 (Ser536) (1:1,000, Bioss, Beijing, China), NF-κB p65 (1:1,000, Proteintech, Wuhan, China), and GAPDH (1:1,000, Bioss, Beijing, China) at 4°C for 16 h. After 3 washes, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:3,000; Proteintech, Wuhan, China).

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Incubation, Western Blot, Control