Journal:

Article Title: Modulation of NF-?B-dependent transcription and cell survival by the SIRT1 deacetylase

doi: 10.1038/sj.emboj.7600244

Figure Lengend Snippet: NAD-dependent HDACs regulate NF-κB transcription. (A) SIRT1 protein level in whole-cell extracts of NSCLC cell lines and normal lung epithelial cell lines (NL-20). (B) NSCLC cells were transiently transfected with the NF-κB-responsive reporter-3xκB luciferase. Cells were left untreated (No Add) or treated overnight with resveratrol (Res, 30 μM), nicotinamide (NAM, 500 μM), splitomicin (Split, 120 μM), or TSA (500 nM). TNFα (10 ng/ml) was added the next day for 8 h before harvesting the cells. All transfections were normalized with CMV-β-galactosidase activity. (C) Cell-based HDAC assays were performed by adding the Fluor de lys substrate alone (No Add) or in combination with resveratrol, nicotinamide, splitomicin, or TSA to the growth medium. Exogenous nicotinamide was added in vitro to untreated cell extracts (No Add+NAM) to ensure that the HDAC activity observed was NAD-dependent. Data represent a typical experiment performed in duplicate, with mean±SD (D) NSCLC cells were co-transfected with the 3xκB luciferase reporter, V5-tagged SIRT1, Myc-tagged HDAC3, or empty vector control (VC). Cells were treated with TNFα for 8 h before harvesting for luciferase activities. Western blot analysis confirmed the expression of V5-tagged SIRT1 and Myc-tagged HDAC3 proteins. (E) NSCLC cells were co-transfected with the 3xκB-luciferase reporter, or expression plasmids encoding SIRT1 protein or vector control. In addition, cells were transfected with p50, RelA/p65 expression vectors, or both.

Article Snippet: NSCLC cells were washed with PBS and incubated with phenol red-free complete media supplemented with 200 μM Fluor de Lys HDAC substrate (BIOMOL) only, or in the presence of resveratrol (30 μM), nicotinamide (500 μM), splitomicin (120 μM), or TSA (500 nM).

Techniques: Transfection, Luciferase, Activity Assay, In Vitro, Plasmid Preparation, Western Blot, Expressing

Journal:

Article Title: Modulation of NF-?B-dependent transcription and cell survival by the SIRT1 deacetylase

doi: 10.1038/sj.emboj.7600244

Figure Lengend Snippet: Resveratrol and SIRT1 inhibit the RelA/p65 transactivation domain by targeting lysine 310. (A) HEK 293T cells were co-transfected with plasmids encoding RelA/p65(1–313) and p300. Whole-cell extracts were immunoprecipitated and incubated with either recombinant SIRT1 or HDAC-defective mutant SIRT1(H363Y) protein in the presence or absence of NAD. (B) HEK 293T cells were co-transfected with expression plasmids as in (A). The transfected cells were left alone or treated with resveratrol (50 μM) or nicotinamide (500 μM) overnight. (C) H460 cells were co-transfected with the Gal4-luciferse reporter and Gal4-p65(286–551) or a mutant Gal4-p65(286–551, K310R). Additionally, cells were transfected with expression plasmids encoding SIRT1 or vector control. Fold repression was calculated where basal transcription values for either Gal4-p65+vector or Gal4-p65(K310R)+vector were normalized to 1.

Article Snippet: NSCLC cells were washed with PBS and incubated with phenol red-free complete media supplemented with 200 μM Fluor de Lys HDAC substrate (BIOMOL) only, or in the presence of resveratrol (30 μM), nicotinamide (500 μM), splitomicin (120 μM), or TSA (500 nM).

Techniques: Transfection, Immunoprecipitation, Incubation, Recombinant, Mutagenesis, Expressing, Plasmid Preparation