Journal: Apoptosis

Article Title: Identification and characterization of the human inhibitor of caspase-activated DNase gene promoter

doi: 10.1007/s10495-008-0223-8

Figure Lengend Snippet: ( a ) Mapping of the transcription start sites by 5′ RACE analysis. Following nested PCR reaction, a PCR product of 100-bp was detected by 1.5% agarose gel electrophoresis (lane 2). Lane 1: no template control; lane M: 100-bp DNA ladder marker. ( b ) The 5′ flanking region of the human ICAD gene. The nucleotide sequence is numbered from the major transcription site, which is indicated by an arrow. Putative binding sites for transcription factors analyzed by the TRANSFAC database and search program were shown with underlines

Article Snippet: DNA transfection of cells with each ICAD -promoter-luciferase construct (1 μg) with an internal control vector pRL-TK (0.1 μg) (Promega) was performed with Trans IT LT-1 (Mirus) during 6-h of incubation.

Techniques: Nested PCR, Agarose Gel Electrophoresis, Control, Marker, Sequencing, Binding Assay

Journal: Apoptosis

Article Title: Identification and characterization of the human inhibitor of caspase-activated DNase gene promoter

doi: 10.1007/s10495-008-0223-8

Figure Lengend Snippet: Human ICAD promoter drives reporter gene expression in transiently transfected Huh-7 cells. Deletion constructs of the upstream region of the ICAD gene linked to the firefly luciferase reporter gene (LUC) are illustrated in the left. Huh-7 cells were cotransfected with a firefly luciferase reporter and a Renilla luciferase internal control (pRL-TK). The relative luciferase activity (RLU) was obtained by normalizing the firefly luciferase activity to the Renilla luciferase activity, and is expressed as a percentage of RLU of pLuc(−936/+71). The average values are shown with standard deviation (SD) ( n = 4 per construct)

Article Snippet: DNA transfection of cells with each ICAD -promoter-luciferase construct (1 μg) with an internal control vector pRL-TK (0.1 μg) (Promega) was performed with Trans IT LT-1 (Mirus) during 6-h of incubation.

Techniques: Gene Expression, Transfection, Construct, Luciferase, Control, Activity Assay, Standard Deviation

Journal: Apoptosis

Article Title: Identification and characterization of the human inhibitor of caspase-activated DNase gene promoter

doi: 10.1007/s10495-008-0223-8

Figure Lengend Snippet: ( a ) ICAD promoter activity after transient expression of c-Myc, N-Myc, USF1 and USF2. Huh-7 cells were cotransfected with either expression vector for c-Myc, N-Myc, USF1 or USF2 driven by the CMV promoter and pLuc(−145/+71), pLuc(−90/+71) or pGL3 basic. Each firefly luciferase reporter plasmid ( n = 4) was cotransfected in cells with pRL-TK for normalization of the reporter activity. RLU is expressed as a percentage of that of pLuc(−145/+71) in the absence of expressing plasmids for Myc and USF. ( b ) Effect of substitution mutation in the E-box element on the ICAD promoter activity. Cells were cotransfected with pLuc(−106/±71) or an E-box-mutant, pLuc(−106/±71)mt and either expression vector for c-Myc or N-Myc. pRL-TK was also used for normalization of the activity. RLU is expressed as a percentage of that of pLuc(−106/±71) without over-expression of Myc

Article Snippet: DNA transfection of cells with each ICAD -promoter-luciferase construct (1 μg) with an internal control vector pRL-TK (0.1 μg) (Promega) was performed with Trans IT LT-1 (Mirus) during 6-h of incubation.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Luciferase, Mutagenesis, Over Expression

Journal: Apoptosis

Article Title: Identification and characterization of the human inhibitor of caspase-activated DNase gene promoter

doi: 10.1007/s10495-008-0223-8

Figure Lengend Snippet: ( a ) An alignment of the sequence around putative Myc binding site of human ICAD promoter and the corresponding part of mouse ICAD sequence. ( b ) Binding of Myc proteins in the endogenous ICAD promoter. Crosslinked chromatins isolated from Huh-7, IMR-32 and GOTO cells were immunoprecipitated with indicated antibodies (c-Myc, N-Myc) or an equivalent amount of mouse IgG. Recovered DNAs were purified and PCR-amplified with primers for nt −272/±71 region or for nt −789/−451 region. Input represents 1% of chromatin sample applied for immunoprecipitation

Article Snippet: DNA transfection of cells with each ICAD -promoter-luciferase construct (1 μg) with an internal control vector pRL-TK (0.1 μg) (Promega) was performed with Trans IT LT-1 (Mirus) during 6-h of incubation.

Techniques: Sequencing, Binding Assay, Isolation, Immunoprecipitation, Purification, Amplification

Journal: Apoptosis

Article Title: Identification and characterization of the human inhibitor of caspase-activated DNase gene promoter

doi: 10.1007/s10495-008-0223-8

Figure Lengend Snippet: Myc-dependent expression of human ICAD. ( a ) Suppression of Myc expression decreases expression of ICAD protein. Huh-7 cells were transiently transfected with Myc siRNA (c-Myc, N-Myc) or the control siRNA (cont). Three days later, the cells were harvested and subjected to Western blotting. ( b ) Ectopic expression of Myc increases expression of ICAD protein. Cells (Huh-7 and mouse 3T3) were transfected with the expression vector for c-Myc or N-Myc, and after 3 days the cells were harvested for Western blotting. ( c ) Comparison of the mRNA expression of c-Myc and ICAD in Huh-7 cells and human oral squamous carcinoma cells (Ca22-9, HSC-2, and HSC-3). Levels of mRNA expression of ICAD and c-Myc were measured by quantitative real-time RT-PCR based on Taq Man chemistry. Results, relative to β-actin mRNA, are depicted as averages with SD ( n = 3)

Article Snippet: DNA transfection of cells with each ICAD -promoter-luciferase construct (1 μg) with an internal control vector pRL-TK (0.1 μg) (Promega) was performed with Trans IT LT-1 (Mirus) during 6-h of incubation.

Techniques: Expressing, Transfection, Control, Western Blot, Plasmid Preparation, Comparison, Quantitative RT-PCR