expression vectors for nf-κb molecules p50, p65, c-rel, p52 and relb  (Addgene inc)

Journal: Acta Pharmaceutica Sinica. B

Article Title: NF- κ B/HDAC1/SREBP1c pathway mediates the inflammation signal in progression of hepatic steatosis

doi: 10.1016/j.apsb.2020.02.005

Figure Lengend Snippet: Impact of P50 knockdown on lipogenic proteins in HepG2 cells. (A) HepG2 cells were electrically transfected with P50 shRNA expression plasmid using cell line nucleofector Kit L, program T031. Pictures were taken using a microscopy with 10 × object lenses under fluorescent light. Scale bar: 200 μm. (B) Oil red staining of triglyceride in transfected HepG2 cells after 0.3 mmol/L oleic acid treatment for 24 h. Relative triglyceride content was quantified in the HepG2 cells by measuring the oil red O amount in the samples. Scale bar: 100 μm. (C) Protein levels of P50, SREBP1, SCD1 and PPAR γ were detected by Western blot in HepG2 cells. In the control group, HepG2 cells were transfected with P50 shRNA plasmid or control vector, and then cultured with DMEM only. In the oleic acid group, transfected cells were treated with 0.3 mmol/L oleic acid in DMEM for 24 h. In the bar figure, each data represents mean ± SE ( n = 3). * P < 0.05, ** P < 0.01.

Article Snippet: HepG2 cells were electrical transfected with mouse P50 -shRNA expression vector (P/N: 9033, L/N: P04122008, Panomics, Redwood, CA, USA) through Amaxa Nucleofector™ II/2b Device, using cell line nucleofector Kit V (VCA-1003, Lonza cologne AG) with the program T-031. shRNA was used at 1–3 μg/well in the 6-well plate.

Techniques: Transfection, shRNA, Expressing, Plasmid Preparation, Microscopy, Staining, Western Blot, Cell Culture

Journal: The Journal of Neuroscience

Article Title: Reduction of Ether-Type Glycerophospholipids, Plasmalogens, by NF-κB Signal Leading to Microglial Activation

doi: 10.1523/JNEUROSCI.3941-15.2017

Figure Lengend Snippet: Suppression of Gnpat expression by NF-κB-induced c-Myc. A, Transfection of p65- and p50-expressing plasmids (1 μg each) in N2A cells (1 × 105 cells/well of 6-well plate) for 48 h increased c-Myc mRNA (left) and protein (right) expression. B, In the same experimental condition, p65 and p50 overexpression reduced Gnpat expression. C, MG6 cells (1 × 105 cells/well of 6 well platees) infected with c-Myc- and Mycn-expressing lentiviruses for 48 h showing the reduction of Gnpat expression compared with control lentivirus infection. D, Luciferase assays in N2A cells showing that the reduction of Gnpat promoter activity by NF-κB (p65 plus p50) (middle panel, third row) and c-Myc (right panel) was canceled by the deletion of Myc-binding sequences from the Gnpat promoter (first and second rows). The data represent six independent experiments. **p < 0.01; ***p < 0.001. E, Western blot data showing the reduction of c-Myc protein expression in MG6 cells infected with the c-Myc sh-RNA-delivering lentivirus for 48 h. F, Gnpat mRNA expression was increased in the c-Myc knockdown cells. G, Reduction of Gnpat expression induced by 6 h treatments with LPS (10 μg/ml), Poly I:C (10 μg/ml), and IL-1β (20 ng/ml) was blocked by c-Myc knockdown in MG6 cells. *p < 0.05; **p < 0.01 (n = 4).

Article Snippet: We thank Stephen Smale for kindly providing the p50 expressing vectors through the Addgene plasmid sharing service, Rhodri E. Jones (Kyushu University) and Ako Niwase for help with English, and the Research Support Center, Graduate School of Medical Sciences, Kyushu University, for technical assistance. .

Techniques: Expressing, Transfection, Over Expression, Infection, Control, Luciferase, Activity Assay, Binding Assay, Western Blot, Knockdown

Journal: The Journal of Neuroscience

Article Title: Reduction of Ether-Type Glycerophospholipids, Plasmalogens, by NF-κB Signal Leading to Microglial Activation

doi: 10.1523/JNEUROSCI.3941-15.2017

Figure Lengend Snippet: Overexpression of p65 and p50 reduces the expression of GNPAT in human cell lines. A, Western blot data showing an increase in c-MYC and decrease in GNPAT protein expression by the transient transfection of p65 and p50 expressing plasmids in SH-SY5Y cells for 48 h. B, Real-time PCR data performed after 48 h of overexpression showing the decrease in GNPAT expression by the overexpression of p65 and p50 in the cells. C, Position of c-MYC-binding sites and the primer locations for the ChIP assays onto the human GNPAT promoter. The number (+1) indicates the transcription start site located within the first exon. D, E, ChIP assays showing the recruitment of c-MYC proteins onto the binding sites of GNPAT promoter in the steady state (D) and after the overexpression of p65 and p50 proteins in the cells for 48 h (E). F, Schematic representing the cloned human GNPAT promoter regions in the luciferase vector. The short form of the promoter (−150 to +183) lacks the putative c-MYC-binding site (C). Promoter assays showed that the overexpression of p65 and p50 proteins could not reduce the promoter activity of the human GNPAT promoter lacking the c-MYC binding site (n = 5).

Article Snippet: We thank Stephen Smale for kindly providing the p50 expressing vectors through the Addgene plasmid sharing service, Rhodri E. Jones (Kyushu University) and Ako Niwase for help with English, and the Research Support Center, Graduate School of Medical Sciences, Kyushu University, for technical assistance. .

Techniques: Over Expression, Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay