Journal: Cell Death & Disease

Article Title: Prolyl-4-hydroxylase 2 enhances hypoxia-induced glioblastoma cell death by regulating the gene expression of hypoxia-inducible factor- α

doi: 10.1038/cddis.2014.295

Figure Lengend Snippet: PHD2 maintains the basal activity of NF κ B. ( a ) U87MG, U138MG and U343MG cells were transfected with non-specific siRNA (Control) or siRNA against PHD2 (PHD2 kd). Forty-eight hours after transfection, p50 was detected by immunoblotting. β -Actin was used as loading control. The results are representative for three independent experiments. ( b ) U87MG and U343MG cells were transfected with water (WT), non-specific siRNA (neg.Control) or siRNA against PHD2 (PHD2 kd). Forty-eight hours after transfection, total mRNA was analyzed for p50 and ribosomal protein L28 expression by qRT-PCR. Normalized p50/L28 ratios are shown. The data are shown as the mean±S.E.M. ( n =3). ** P <0.01. ( c ) U87MG cells were co-transfected with NF κ B responsive firefly luciferase plasmids and renilla luciferase plasmids. Six hours later, cells were transfected with siRNA against PHD2 and incubated further for 48 h. Firefly luciferase activities (FL) were normalized to Renilla luciferase activities (RL). The data are shown as the mean±S.E.M. ( n =3). ** P <0.01

Article Snippet: Cells were cultured to 60% confluence and co-transfected with a firefly luciferase plasmid containing five copies of NF κ B binding sites (Promega, Mannheim, Germany) and a Renilla luciferase plasmid as transfection efficiency control.

Techniques: Activity Assay, Transfection, Control, Western Blot, Expressing, Quantitative RT-PCR, Luciferase, Incubation

Journal: Current Research in Toxicology

Article Title: Wheatgrass inhibits the lipopolysaccharide-stimulated inflammatory effect in RAW 264.7 macrophages

doi: 10.1016/j.crtox.2021.02.005

Figure Lengend Snippet: Effect of wheatgrass extract on the transactivation of NF-κB promoter in LPS-stimulated RAW 264.7 cells. Raw 264.7 cells were transfected with pNFκB-Luc and β-gal plasmids and then exposed to 1 µg/ml of LPS in the presence or absence of W5 (A) and W7 (B) for 12 h followed by estimation of luciferase activities. Results are mean ± SEM of three independent experiments. * and # indicate p < 0.05 as compared to vehicle-treated control and LPS-stimulated cells, respectively. W5, 5-day old wheatgrass methanol extract; W7, 7-day old wheatgrass methanol extract; LPS, lipopolysaccharide.

Article Snippet: For this assay, RAW 264.7 cells were co-transfected with a p-NF-κB-Luc reporter, containing mammalian NF-κB response element in its promoter region, and β-gal plasmids (both procured from Promega Corporation, Madison, WI, USA), where the latter was used as an internal control.

Techniques: Transfection, Luciferase

Journal: PLoS ONE

Article Title: Gadd153 and NF-κB Crosstalk Regulates 27-Hydroxycholesterol-Induced Increase in BACE1 and β-Amyloid Production in Human Neuroblastoma SH-SY5Y Cells

doi: 10.1371/journal.pone.0070773

Figure Lengend Snippet: (a,b) 27-OHC significantly increases the levels of the p65 and the p50 subunits of the NF-κB in the nuclear homogenates, and (c–e) increases the protein and mRNA levels of BACE1; treatment with the NF-κB inhibitor sc514 attenuates the 27-OHC-induced increase in protein and mRNA expression of BACE1. (f) ChIP analysis shows that 27-OHC increases the binding of NF-κB in the BACE1 promoter region. (g) Dual luciferase assay demonstrates that 27-OHC increases the NF-κB transcriptional activity as measured by a significant increase in NF-κB reporter activity. (h) Dual luciferase assay demonstrates that 27-OHC significantly increases the BACE1 promoter activity, while the NF-κB inhibitor sc514 decreases the 27-OHC-induced increase in BACE1 promoter activity. Data is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). *p<0.05, **p<0.01, and ***p<0.001 versus control, † p<0.05, †† p<0.01, and ††† p<0.001 versus 27-OHC.

Article Snippet: 27-OHC was purchased from Medical Isotopes (Pelham, NH), the NF-κB inhibitor sc514 from Tocris Bioscience (Ellisville, MO), the reporter constructs encoding NF-κB response elements conjugated to the firefly luciferase gene from SA Biosciences (Frederick, MD), and the human BACE1 promoter construct conjugated to the firefly luciferase gene was purchased from SwitchGear Genomics (Menlo Park, CA).

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Cell Culture, Control

Journal: PLoS ONE

Article Title: Gadd153 and NF-κB Crosstalk Regulates 27-Hydroxycholesterol-Induced Increase in BACE1 and β-Amyloid Production in Human Neuroblastoma SH-SY5Y Cells

doi: 10.1371/journal.pone.0070773

Figure Lengend Snippet: (a–c) siRNA to gadd153 mitigates the 27-OHC-induced increase in BACE1 protein levels and mRNA expression. (d) EMSA shows that 27-OHC increases the binding of NF-κB to the exogenous DNA sequence that corresponds to the NF-κB binding site in the BACE1 promoter; silencing gadd153 expression reduces the increase in the binding of NF-κB. (e) ChIP analysis shows that siRNA to gadd153 decreases the 27-OHC-induced increase in binding of NF-κB to the BACE1 promoter. (f,g) Dual luciferase assay demonstrates that siRNA to gadd153 decreases the 27-OHC-induced increase in NF-κB transcriptional activity and BACE1 promoter activity. (h) Levels of gadd153 are detectable in hippocampus of 3, 6, and 12 month-old 3xTg-AD, while NF-κB levels are detectable in hippocampus of 6 and 12month-old, and BACE1 levels increasing only in 12 month-old 3xTg-AD. Data in a-g is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). *p<0.05, **p<0.01, and ***p<0.001 versus control, †† p<0.01 and ††† p<0.001 versus 27-OHC.

Article Snippet: 27-OHC was purchased from Medical Isotopes (Pelham, NH), the NF-κB inhibitor sc514 from Tocris Bioscience (Ellisville, MO), the reporter constructs encoding NF-κB response elements conjugated to the firefly luciferase gene from SA Biosciences (Frederick, MD), and the human BACE1 promoter construct conjugated to the firefly luciferase gene was purchased from SwitchGear Genomics (Menlo Park, CA).

Techniques: Expressing, Binding Assay, Sequencing, Luciferase, Activity Assay, Cell Culture, Control