Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of a novel pro-apopotic function of NF-κB in the DNA damage response

doi: 10.1111/j.1582-4934.2009.00888.x

Figure Lengend Snippet: Doxorubicin-induced NF-κB activation enhances DNA damage and apoptosis in A172 glioblastoma cells. (A), Ectopic expression of IκBα-SR. A172 glioblastoma cells were stably transduced with a control vector or a vector containing IκBα-SR. Protein expression of wild-type IκBα and mutant IκBα-SR was determined by Western blot analysis. β-actin served as loading control. (B), Inhibition of NF-κB DNA binding by IκBα-SR. NF-κB DNA binding was assessed by EMSA in nuclear extracts of A172 cells transduced with control vector or a vector containing IκBα-SR that were left untreated or were treated with 0.8 μg/ml Doxorubicin for 6 hrs or 10 ng/ml TNFα for 1 hr. (C), Inhibition of NF-κB transcriptional activity by IκBα-SR. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were transiently transfected with firefly and renilla luciferase gene constructs, treated for 6 hrs with 10 ng/ml TNFα or for 24 hrs with 0.8 μg/ml Doxorubicin and analysed by dual luciferase assay for induction of NF-κB transcriptional activity. Fold increase in luciferase activity relative to unstimulated control is shown. (D), Enhancement of TNFα-induced apoptosis by NF-κB inhibition. A172 cells transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were left untreated (–TNFα) or were treated with 50 ng/ml TNFα for 48 hrs (+TNFα). Apoptosis was determined by FACS analysis of DNA-fragmentation of propidium iodide stained nuclei. (E), NF-κB promotes Doxorubicin-induced DNA damage. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, DNA damage was assayed by Comet assay and is displayed as Olive Tail Moment. (F), NF-κB promotes Doxorubicin-induced apoptosis. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, apoptosis was determined by FACS analysis of DNA-fragmentation of propidium-iodide stained nuclei. Median (E) or mean (C, D and F) + S.D. of three independent experiments are shown; * P < 0.05 and # P < 0.001 comparing IκBα-SR versus control.

Article Snippet: Human glioblastoma cell lines U87MG, T98G and A172 were obtained from ATCC and grown in DMEM medium (Invitrogen, Karlsruhe, Germany) supplemented with 1% penicillin/streptomycin, 1 mmol/l L-glutamine (both from Invitrogen), 10% foetal calf serum and 25 mmol/l HEPES (both from Biochrom AG, Berlin, Germany).

Techniques: Activation Assay, Expressing, Stable Transfection, Transduction, Control, Plasmid Preparation, Mutagenesis, Western Blot, Inhibition, Binding Assay, Activity Assay, Transfection, Luciferase, Construct, Staining, Single Cell Gel Electrophoresis

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Intestinal Lipid Handling

doi: 10.1161/atvbaha.113.302993

Figure Lengend Snippet: Figure 4. Intestinal lipid synthesis and lipoprotein biogenesis in the small intestine of insulin-sensitive and insulin-resistant obese subjects. Tissue homogenates were analyzed by immunoblotting for the phosphorylation of 5′-adenosine monophosphate–activated protein kinase α (AMPKα) at Thr172 (A), AMPKα (B), acetyl-CoA carboxylase (ACC) at Ser79 (C), and ACC (D). Densitometric analyses of protein expres- sion were normalized for protein expression levels of β-actin. Intestinal de novo lipogenesis rates were measured by the incorporation of [1-14C]-acetic acid for 3 h (E). Data are expressed as nanomoles of acetic acid incorporated by a milligram of protein (insulin-sensitive, n=7; insulin-resistant, n=9). Apolipoprotein (Apo) B-48 synthesis by intestinal explants was evaluated by the incorporation of [35S]-methionine in immunopurified Apo B-48 resolved on SDS-PAGE acrylamide gel (F). Data are expressed as DPM of [35S]-methionine incorporated by a milligram of protein (insulin-sensitive, n=7; insulin-resistant, n=9). Triglyceride (TG)-rich lipoprotein (TRL) production by intestinal explants was evaluated by the incorporation of [1-14C]-oleic acid in TRL isolated by ultracentrifugation (G). Data are expressed as DPM of [1-14C]-oleic acid incorporated by a milligram of protein (insulin-sensitive, n=7; insulin-resistant, n=9). Data are mean±SEM (*P<0.05).

Article Snippet: The following antibodies (directed against human) and dilutions (1:1000 unless otherwise specified) were employed: mouse anti-β-actin (1:40000, Sigma Aldrich, St. Louis, USA); anti-Akt Veilleux et al., Data Supplements, ATVB/2013/302993D, Page 2 (#ab32902) and anti-TNF-α (#ab66579) from Abcam (Cambridge, USA); antiintestinal-fatty acid binding protein (I-FABP) and anti-liver-fatty acid binding protein (L-FABP) antibodies were raised in rabbits after injection of recombinant proteins;3 anti-microsomal transfer protein (MTP) was kindly provided by John Wetterau and Harris Jamil (Bristol-Myers Squibb Research Institute, USA);4 antiSAR-1B (1:2000) was kindly provided by Randy Schekman (University of California, USA);4 anti-PCSK9 was kindly provided by Geneviève Dubuc and Jean Davignon (Clinical Research Institute of Montreal, Canada);5 anti-nuclear factor kappa B (NF-kB) p65 subunit (sc-372G) and anti-I-KappaB-alpha (IKB-α) (sc-1643) were obtained from Santa-Cruz Biotechnology (Santa-Cruz, USA); anti-phospho-Akt Ser473 (#9271), anti-phospho-AMPKα Thr172 (#40H9), antiAMPKα (#2532), anti-phospho-p38 MAPK Thr180/Tyr182 (#4631), anti-p38 MAPK (#9212), anti-phospho-JNK Thr183/Tyr185 (#9251), anti-JNK (#9252), anti-phospho-Acetyl-CoA Carboxylase (ACC) Ser79 (#3661) and anti-ACC (#3662) were obtained from Cell Signaling Technology (Boston, USA).

Techniques: Western Blot, Phospho-proteomics, Expressing, SDS Page, Acrylamide Gel Assay, Isolation