Journal: Journal of dairy science

Article Title: Sodium butyrate reduces bovine mammary epithelial cell inflammatory responses induced by exogenous lipopolysaccharide, by inactivating NF-κB signaling.

doi: 10.3168/jds.2020-18189

Figure Lengend Snippet: Figure 5. Effects of sodium butyrate (SB) on NF-κB signaling pathway. Bovine mammary epithelial cells (MAC-T) were treated with 1 µg/mL LPS for 6 h; with 0.5 mM SB for 18 h; or with 0.5 mM SB for 18 h, followed by a challenge with 1 µg/mL LPS for an additional 6 h. (A) Western blot analysis of p-IκB α, IκB α, p-p65, and p65. (B) Relative protein expression levels of p-IκB α to IκB α. (C) Relative protein expression levels of p-p65 to p65. Data from the control group were used to normalize the data of each treatment group. Comparisons among groups were calculated using a one-way ANOVA with subsequent Bonferroni correction. Data presented are mean ± SEM. Different lowercase letters in bar charts indicate significant differences (P < 0.05).

Article Snippet: Membranes were then incubated with antibodies against phosphorylatd-IκB α (p-IκB α; sc-271980, Santa Cruz Biotechnology; 1:5,000), IκB α (sc-166588, Santa Cruz Biotechnology; 1:1,000), NFκB p65 (sc-8008, Santa Cruz Biotechnology; 1:1,000), phosphorylatd-NF-κB p65 (p-NF-κB p65; sc-166748, Santa Cruz Biotechnology; 1:1,000), acetyl-H3 (8173, Cell Signaling Technology, Danvers, MA; 1:1,000), histone H3 (4499, Cell Signaling Technology; 1:1,000), and GAPDH (ab8245, Abcam, Cambridge, MA; 1:5,000) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (Boster, Wuhan, China) for 45 min at room temperature.

Techniques: Western Blot, Expressing, Control

Journal: Cancer Biology & Therapy

Article Title: The NFκB inhibitor, SN50, induces differentiation of glioma stem cells and suppresses their oncogenic phenotype

doi: 10.4161/cbt.28158

Figure Lengend Snippet: Figure 3. Effects of silencing of NFκB/p50 expression on differentiation of GSCs. (A) GSC11 cells were dissociated, suspended in serum-free DMEM medium in the absence of B-27, EGF, and β-FGF, and then were infected with an NFκB/p50-targeted shRNA lentiviral particles for 48 h in the presence of polybrene (5 μg/mL). Forty-eight hours later, (A) the cells were observed under a phase contrast microscopy (200× magnification). **P < 0.01, shNFκB/p50 vs. shNT control; (B) cell lysates were prepared and subjected to western blot analysis of CD133, Sox2, MAP-2, and p50. β-actin was used as a loading control.

Article Snippet: GSC11 cells were dissociated, plated into 6-well plates, and then infected with an NFκB/p50-targeted shRNA lentiviral particles (Santa Cruz Biotechnology, Inc.) in the presence of 5 μg/mL of polybrene (Sigma-Aldrich) for 24 h. The cells were then selected with puromycin.

Techniques: Expressing, Infection, shRNA, Microscopy, Western Blot

Journal: Archives of toxicology

Article Title: MicroRNA-942 mediates hepatic stellate cell activation by regulating BAMBI expression in human liver fibrosis

doi: 10.1007/s00204-018-2278-9

Figure Lengend Snippet: NF-κB p50 mediate miR-942 upregulation in response to LPS. a miR-942 expression in liver cell types after 24 h culture as measured by real-time PCR. Bars show mean ± SD, n = 3 per group. hHSC human primary hepatic stellate cells, Hepa hepatocytes, KCs Kupffer cells, LSECs liver sinusoidal endothelial cells. b miR-942 expression in hHSCs and c LX2 in response to LPS and TGF-β1 stimulation. The cells were stimulated with 5 ng/ml TGF-β1 for 48 h, followed by a 4 h stimulation with 100 ng/ml LPS. Bars show mean ± SD, n = 3 per group of three independent experiments. d miR-942 expression in hHSCs and e LX2 after 4 h stimulation with LPS (100 ng/ml). Where Bay11–7082 is indicated, hHSCs were pre-treated with 1 μM of the inhibitor for 2 h before LPS treatment. Un untreated. Data represent mean ± SD of three independent experiments. f Real-time PCR data following siRNA-mediated knockdown of p50 and (g) p65. On the upper part of the figures: immunoblots showing knockdown efficacy 48 h after transfection. LX2 cells were stimulated with 100 ng/ml LPS for 4 h. Un untreated. Bars show mean ± SD, n = 3 per group of three independent experiments. h Sequence of the NF-κB binding site at position − 215 of the miR-942 promoter as predicted with bioinformatics platforms. Below: a ChIP analysis showing the effect of LPS (100 ng/ml for 1 h) on p50 recruitment to the NF-κB binding site of the human miR-942 promoter. Following treatment with LPS, cells were fixed and soluble chromatin was immunoprecipitated using anti-p50 or control abs (normal mouse IgG), as indicated. The miR-942 promoter fragment containing the – 215-binding site was amplified by PCR. Un untreated

Article Snippet: Transfection of siRNA targeting NF-κB p50, Smad2/3, BAMBI, as well as non-targeting (scrambled) control was carried out according to manufacturer’s protocol (Santa Cruz Biotech.Inc, CA, USA), using Lipofectamine LTX as transfection reagent.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Knockdown, Western Blot, Transfection, Sequencing, Binding Assay, Immunoprecipitation, Control, Amplification

Journal: Archives of toxicology

Article Title: MicroRNA-942 mediates hepatic stellate cell activation by regulating BAMBI expression in human liver fibrosis

doi: 10.1007/s00204-018-2278-9

Figure Lengend Snippet: miR-942 and BAMBI correlation with fibrotic stage and proposed mechanism in HSC activation. a Pearson correlation analysis indicating an inverse relationship between miR-942 and BAMBI mRNA levels. r=correlation coefficient, statistical significance – P < 0.05. b Pearson correlation analysis indicating direct association between high miR-942 expression and fibrotic stage. c Proposed mechanism of miR-942-mediated liver fibrosis. Upon stimulation with TGF-β1 and LPS, Smad2/3 and NF-κB p50 respectively bind to miR-942 promoter. This leads to the induction of miR-942 expression, the subsequent downregulation of BAMBI, and HSC activation. HSC activation then orchestrates fibrosis

Article Snippet: Transfection of siRNA targeting NF-κB p50, Smad2/3, BAMBI, as well as non-targeting (scrambled) control was carried out according to manufacturer’s protocol (Santa Cruz Biotech.Inc, CA, USA), using Lipofectamine LTX as transfection reagent.

Techniques: Activation Assay, Expressing

Journal: Cancer Biology & Therapy

Article Title: The NFκB inhibitor, SN50, induces differentiation of glioma stem cells and suppresses their oncogenic phenotype

doi: 10.4161/cbt.28158

Figure Lengend Snippet: Figure 3. Effects of silencing of NFκB/p50 expression on differentiation of GSCs. (A) GSC11 cells were dissociated, suspended in serum-free DMEM medium in the absence of B-27, EGF, and β-FGF, and then were infected with an NFκB/p50-targeted shRNA lentiviral particles for 48 h in the presence of polybrene (5 μg/mL). Forty-eight hours later, (A) the cells were observed under a phase contrast microscopy (200× magnification). **P < 0.01, shNFκB/p50 vs. shNT control; (B) cell lysates were prepared and subjected to western blot analysis of CD133, Sox2, MAP-2, and p50. β-actin was used as a loading control.

Article Snippet: GSC11 cells were dissociated, plated into 6-well plates, and then infected with an NFκB/p50-targeted shRNA lentiviral particles (Santa Cruz Biotechnology, Inc.) in the presence of 5 μg/mL of polybrene (Sigma-Aldrich) for 24 h. The cells were then selected with puromycin.

Techniques: Expressing, Infection, shRNA, Microscopy, Control, Western Blot