Journal: Journal of Lipid Research

Article Title: Control of ACAT2 liver expression by HNF1

doi: 10.1194/jlr.m400450-jlr200

Figure Lengend Snippet: Fig. 2. Effects of deletions of hepatic nuclear factor 1 (HNF1) and CCAAT/enhancer binding protein (C/EBP) elements in the human ACAT2 gene on hepatocyte-specific expression. pC/EBP mutant, pHNF1 mutant, and pHNF1 and C/EBP mutant con- structs (A), carrying deletions for the putative C/EBP, the putative HNF1, and for both putative binding sites in the p1044 construct, respectively, were transiently transfected into HuH7 cells (B) and HepG2 cells (C). D: pHNF1 mutant p1305 construct, carrying a deletion for the putative HNF1 binding site in the full-length pro- moter construct, was transiently transfected into HepG2. pSV- -galactosidase (-Gal) control vector served as an internal control of transfection efficiency. The empty (pGL3) reporter vector served as a baseline control. Luciferase and -galactosidase activities were measured in cell lysates 48 h after transfection. Luciferase activity was corrected by -galactosidase activity. All transfections were per- formed in five replicates, and data represent means SEM.

Article Snippet: antibody (catalog number Sc-6547X; Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of HNF1 antibody (catalog number Sc-22840X; Santa Cruz Biotechnology) was added to the binding reaction mixture.

Techniques: Binding Assay, Expressing, Mutagenesis, Construct, Transfection, Control, Plasmid Preparation, Luciferase, Activity Assay

Journal: Journal of Lipid Research

Article Title: Control of ACAT2 liver expression by HNF1

doi: 10.1194/jlr.m400450-jlr200

Figure Lengend Snippet: Fig. 3. Functionality of the HNF1 binding site in the human ACAT2 gene. Nuclear extracts, prepared from HuH7 cells, were incubated with 32P-labeled double-stranded probes with or without a mutation for the HNF1 binding site and resolved on a nondenatured (5%, w/v) acrylamide gel. For competition assays, a 100- fold molar excess of unlabeled probe was added to the binding reaction mixture, and for the supershift as- says, 2 g of HNF1 or HNF1 antibodies was added. Lanes 1 and 2, binding reaction between 10 and 20 g nuclear extracts, respectively, and 32P-labeled HNF1 probe. Lane 3, competition between labeled and unla- beled HNF1 probe. Lane 4, competition between labeled HNF1 probe and unlabeled mutated HNF1 probe. Lane 5, supershift reaction between labeled HNF1 probe and 2 g of HNF1 antibody. Lane 6, supershift re- action between labeled HNF1 probe and 2 g of HNF1 antibody. Lanes 7 and 8, binding reaction between 10 and 20 g nuclear extracts, respectively, and labeled mutated HNF1 probe. Lane 9, 10 g of BSA and la- beled HNF1 probe, serving as a negative control (Neg. Ctrl).

Article Snippet: antibody (catalog number Sc-6547X; Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of HNF1 antibody (catalog number Sc-22840X; Santa Cruz Biotechnology) was added to the binding reaction mixture.

Techniques: Binding Assay, Incubation, Labeling, Mutagenesis, Acrylamide Gel Assay, Negative Control

Journal: Journal of Lipid Research

Article Title: Control of ACAT2 liver expression by HNF1

doi: 10.1194/jlr.m400450-jlr200

Figure Lengend Snippet: Fig. 4. In vivo association of HNF1 and HNF1 with the human ACAT2 promoter in liver. Soluble chromatin was prepared from 200 mg of human liver and immunoprecipitated with specific anti- bodies against HNF1, HNF1, or IgG and amplified using real- time RT-PCR, conducted in triplicate. The IgG antibody was used as a baseline control and to compare the relative fold enrichment of the ACAT2 promoter by the specific DNA fragments. Before immu- noprecipitations, a small aliquot of chromatin was saved and used as an input control. ChIP, chromatin immunoprecipitation.

Article Snippet: antibody (catalog number Sc-6547X; Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of HNF1 antibody (catalog number Sc-22840X; Santa Cruz Biotechnology) was added to the binding reaction mixture.

Techniques: In Vivo, Immunoprecipitation, Quantitative RT-PCR, Control, Chromatin Immunoprecipitation

Journal: Journal of Lipid Research

Article Title: Control of ACAT2 liver expression by HNF1

doi: 10.1194/jlr.m400450-jlr200

Figure Lengend Snippet: Fig. 5. Importance of the HNF1 binding site for hepatocyte-spe- cific expression. The p1044 construct (A) or the pHNF1 mu- tant construct (B) carrying a deletion for the putative HNF1 bind- ing site, and 0.1 or 0.5 g of HNF1 and/or HNF1 expression vector, was cotransfected into HuH7 cells. pSV--galactosidase (-Gal) control vector served as an internal control of transfection efficiency. The empty (pGL3) reporter vector served as a baseline control. Luciferase and -galactosidase activities were measured in cell lysates 48 h after transfection. Luciferase activity was corrected by -galactosidase activity. All transfections were performed in five replicates, and data represent means SEM.

Article Snippet: antibody (catalog number Sc-6547X; Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of HNF1 antibody (catalog number Sc-22840X; Santa Cruz Biotechnology) was added to the binding reaction mixture.

Techniques: Binding Assay, Expressing, Construct, Plasmid Preparation, Control, Transfection, Luciferase, Activity Assay

Journal: Journal of Lipid Research

Article Title: Control of ACAT2 liver expression by HNF1

doi: 10.1194/jlr.m400450-jlr200

Figure Lengend Snippet: Fig. 6. Influence of HNF1 and HNF1 overexpression on hu- man ACAT2 promoter activity in different cells. The human ACAT2 full-length promoter construct (p1305), along with 0.1 or 0.2 g of HNF1 and/or HNF1 expression vector, was transiently cotransfected into HepG2 cells (A), into HuH7 cells (B), or into HEK293 cells (C). pSV--galactosidase (-Gal) control vector served as an internal control of transfection efficiency. The empty (pGL3) reporter vector served as a baseline control. Luciferase and -galactosidase activities were measured in cell lysates 48 h after transfection. Luciferase activity was corrected by -galactosidase ac- tivity. All transfections were performed in five replicates, and data represent means SEM.

Article Snippet: antibody (catalog number Sc-6547X; Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of HNF1 antibody (catalog number Sc-22840X; Santa Cruz Biotechnology) was added to the binding reaction mixture.

Techniques: Over Expression, Activity Assay, Construct, Expressing, Plasmid Preparation, Control, Transfection, Luciferase

Journal: Journal of Lipid Research

Article Title: Control of ACAT2 liver expression by HNF1

doi: 10.1194/jlr.m400450-jlr200

Figure Lengend Snippet: Fig. 7. Alignment between human and mouse 5 flanking regions of the ACAT2 gene. Shaded letters show base homology. aataaa, poly-A sequence of the IGFBP-6 gene; Cdx-2, caudal-related homeodomain protein-2; framed HNF1, sequence for the HNF1 responsible for the hepatocyte-specific expression of the ACAT2 gene in humans; (HNF1), cis-element for HNF1 present only in the mouse sequence. The longer underlined se- quence shows part of the exon sequence.

Article Snippet: antibody (catalog number Sc-6547X; Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of HNF1 antibody (catalog number Sc-22840X; Santa Cruz Biotechnology) was added to the binding reaction mixture.

Techniques: Sequencing, Expressing

Journal: Pharmaceutics

Article Title: Olaparib Conjugates with Selenopheno[3,2- c ]quinolinone Inhibit PARP1 and Reverse ABCB1-Related Multidrug Resistance

doi: 10.3390/pharmaceutics14122571

Figure Lengend Snippet: Cytotoxicity of 5a – c on the MES-SA/Dx-5 cell line.

Article Snippet: MES-SA (Human uterine sarcoma, ATCC ® CRL-1976TM) and H9C2 (rat cardiomyocytes, ATCC CRL-1446TM), MCF-7 (human adenocarcinoma, ATCC HTB-22TM), and HCC1937 (human breast carcinoma, ATCC CRL-2336TM) cell lines were obtained from American Type Culture Collection, and the MES-SA/Dx-5 (doxorubicin resistant human uterine sarcoma with high levels of MDR1 mRNA and P-glycoprotein, ECACC 95051031-1VL) cell line was obtained from the European Collection of Authenticated Cell Cultures.

Techniques: