Journal: Oncotarget

Article Title: GATA3 and APOBEC3B are prognostic markers in adrenocortical carcinoma and APOBEC3B is directly transcriptionally regulated by GATA3

doi: 10.18632/oncotarget.27703

Figure Lengend Snippet: ( A ) TaqMan quantitative real-time PCR of APOBEC3B in ACCs in our cohort of adrenocortical tissue samples. Normal, n = 21; Benign, n = 69; ACC, n = 38. ( B ) APOBEC3B expression in publicly available microarray data from the GEO database, GSE10927. Normal, n = 10; ACA, n = 22; ACC, n = 33. The data were analyzed through GEO2R online software tool and all the data points were shown in Box-whiskers plots ( C ) APOBEC3B expression in another publicly available data from EMBL-EBI, E-TABM-311. Normal n = 4; ACA, n = 58; ACC, n = 34. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. The author submitted normalized expression data were collected directly and all the data points were shown in Box-whiskers plots ( D ) Representative immunofluorescent staining images for APOBEC3B expression in adrenocortical tissues. DAPI is used to stain the cell nuclei for reference (magnification, 40×). TaqMan gene expression ( E ) and protein expression ( F ) of APOBEC3B in three adrenocortical carcinoma cell lines, BD140A, H295R, SW13, and normal human embryonic kidney cell line, HEK293. We used H295R and BD140 adrenocortical carcinoma cell lines for subsequent experiments as they are more representative of adrenocortical carcinomas.

Article Snippet: Four lentiviral psi-LVRU6P-01, psi-LVRU6P-02, psi-LVRU6P-03 and psi-LVRU6P-04 vectors (catalog # CS-HSH022939-LVRU6P-01 to LVRU6P-04) with U6 promoter expressing shRNAs targeting APOBEC3B and one shRNA scrambled control clone for psi-LVRU6P (catalog # CSHCTR001-LVRU6P) were obtained from GeneCopoeia (Rockville, MD, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Microarray, Software, Staining, Gene Expression

Journal: Oncotarget

Article Title: GATA3 and APOBEC3B are prognostic markers in adrenocortical carcinoma and APOBEC3B is directly transcriptionally regulated by GATA3

doi: 10.18632/oncotarget.27703

Figure Lengend Snippet: Knockdown of APOBEC3B gene expression over time using two siRNAs targeting (A3Bsi1, A3Bsi2) both isoforms in BD140A ( A ) and H295R ( B ) cell lines by TaqMan quantitative real-time PCR. Percentage expression has been normalized to scrambled siRNA which was used as a negative control. The cell proliferation growth curve after transient transfection of two siRNAs in BD140A ( C ) and H295R ( D ) cell lines as measured by CyQuant assay. * P < 0.05; *** P < 0.001. Cell cycle analysis in BD140A ( E ) and H295R ( F ) cell lines after transient knockdown of APOBEC3B for 72 hours. The proportion of cells in each phase is represented as a percentage next to their corresponding peaks.

Article Snippet: Four lentiviral psi-LVRU6P-01, psi-LVRU6P-02, psi-LVRU6P-03 and psi-LVRU6P-04 vectors (catalog # CS-HSH022939-LVRU6P-01 to LVRU6P-04) with U6 promoter expressing shRNAs targeting APOBEC3B and one shRNA scrambled control clone for psi-LVRU6P (catalog # CSHCTR001-LVRU6P) were obtained from GeneCopoeia (Rockville, MD, USA).

Techniques: Knockdown, Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Negative Control, Transfection, CyQUANT Assay, Cell Cycle Assay

Journal: Oncotarget

Article Title: GATA3 and APOBEC3B are prognostic markers in adrenocortical carcinoma and APOBEC3B is directly transcriptionally regulated by GATA3

doi: 10.18632/oncotarget.27703

Figure Lengend Snippet: ( A ) Representative co-immunofluorescent staining images of APOBEC3B and γH2AX in normal, benign adrenocortical tumors and adrenocortical carcinoma. DAPI was used to stain the cell nuclei as a reference. (magnification, 40×) ( B ) The table represents the list and type of mutations within the TP53 gene that was screened in adrenocortical tumors using Sanger DNA sequencing. ( C ) The adrenocortical tumors were stratified into two groups (high and low expression levels) based on the median gene expression level of APOBEC3B and the average number of observed TP53 mutations in each patient was represented. The y-axis represents the sequencing data obtained from both forward and reverse strands for a given mutation. Error bar represents the standard error mean (SEM).

Article Snippet: Four lentiviral psi-LVRU6P-01, psi-LVRU6P-02, psi-LVRU6P-03 and psi-LVRU6P-04 vectors (catalog # CS-HSH022939-LVRU6P-01 to LVRU6P-04) with U6 promoter expressing shRNAs targeting APOBEC3B and one shRNA scrambled control clone for psi-LVRU6P (catalog # CSHCTR001-LVRU6P) were obtained from GeneCopoeia (Rockville, MD, USA).

Techniques: Staining, DNA Sequencing, Expressing, Gene Expression, Sequencing, Mutagenesis

Journal: Oncotarget

Article Title: GATA3 and APOBEC3B are prognostic markers in adrenocortical carcinoma and APOBEC3B is directly transcriptionally regulated by GATA3

doi: 10.18632/oncotarget.27703

Figure Lengend Snippet: ( A ) Genome-wide CGH array representing cumulative copy number gain or loss for all chromosomes in adrenocortical carcinoma samples were dichotomized by the median gene expression level of APOBEC3B in the tumor samples. Red indicates the copy number loss while blue represents the copy number gain in a given chromosome. The columns represent each chromosome. Overall patient survival dichotomized based on the median gene expression level of APOBEC3B from two publicly available datasets; E-TABM-311 ( B ), GSE19776, ( C ) our cohort, ( D ) and the TCGA adrenocortical cancer data ( E ).

Article Snippet: Four lentiviral psi-LVRU6P-01, psi-LVRU6P-02, psi-LVRU6P-03 and psi-LVRU6P-04 vectors (catalog # CS-HSH022939-LVRU6P-01 to LVRU6P-04) with U6 promoter expressing shRNAs targeting APOBEC3B and one shRNA scrambled control clone for psi-LVRU6P (catalog # CSHCTR001-LVRU6P) were obtained from GeneCopoeia (Rockville, MD, USA).

Techniques: Genome Wide, Gene Expression

Journal: Oncotarget

Article Title: GATA3 and APOBEC3B are prognostic markers in adrenocortical carcinoma and APOBEC3B is directly transcriptionally regulated by GATA3

doi: 10.18632/oncotarget.27703

Figure Lengend Snippet: ( A ) APOBEC3B gene expression (log2 fold change) analyzed in a functional screen of 92 cancer-associated transcription factors. ( B ) Independent validation of four candidates by TaqMan gene expression in H295R cells. Luciferase reporter assay under the control of APOBEC3B gene promoter after transient knockdown of GATA3 transcription factor with two different siRNAs in BD140A ( C ) and H295R ( D ) cell lines. ( E ) Overall patient survival dichotomized based on the median gene expression level of GATA3 from the TCGA adrenocortical cancer dataset.

Article Snippet: Four lentiviral psi-LVRU6P-01, psi-LVRU6P-02, psi-LVRU6P-03 and psi-LVRU6P-04 vectors (catalog # CS-HSH022939-LVRU6P-01 to LVRU6P-04) with U6 promoter expressing shRNAs targeting APOBEC3B and one shRNA scrambled control clone for psi-LVRU6P (catalog # CSHCTR001-LVRU6P) were obtained from GeneCopoeia (Rockville, MD, USA).

Techniques: Gene Expression, Functional Assay, Biomarker Discovery, Luciferase, Reporter Assay, Control, Knockdown

Journal: Oncotarget

Article Title: GATA3 and APOBEC3B are prognostic markers in adrenocortical carcinoma and APOBEC3B is directly transcriptionally regulated by GATA3

doi: 10.18632/oncotarget.27703

Figure Lengend Snippet: APOBEC3B TCGA OS multivariate analysis

Article Snippet: Four lentiviral psi-LVRU6P-01, psi-LVRU6P-02, psi-LVRU6P-03 and psi-LVRU6P-04 vectors (catalog # CS-HSH022939-LVRU6P-01 to LVRU6P-04) with U6 promoter expressing shRNAs targeting APOBEC3B and one shRNA scrambled control clone for psi-LVRU6P (catalog # CSHCTR001-LVRU6P) were obtained from GeneCopoeia (Rockville, MD, USA).

Techniques:

Journal: Oncotarget

Article Title: GATA3 and APOBEC3B are prognostic markers in adrenocortical carcinoma and APOBEC3B is directly transcriptionally regulated by GATA3

doi: 10.18632/oncotarget.27703

Figure Lengend Snippet: ( A ) Schematic representation of the GATA3 binding sites on the APOBEC3B promoter region. Arrows indicate the primer probes used for ChiP-PCR; TSS, transcription start site. ( B ) Agarose gel electrophoresis of SYBR green PCR products produced from site A and site B specific primer probes of APOBEC3B promoter region after chromatin immunoprecipitation (ChIP) with GATA3 antibody. Primers were also designed for Exon 5 of APOBEC3B where there are no putative binding sites for GATA3 as a negative control. RNApolII was used as a positive control for ChiP and housekeeping gene GAPDH was used for validation. Previously published GATA3 binding target, GCMB gene specific primers was used as a positive control for GATA3 ChiP antibody . A reaction mixture without template was used as a negative control for PCR. Fold enrichment of GATA3 binding sites A and B after ChIP with GATA3 antibody relative to immunoglobulin (IgG) antibody in BD140A ( C ) and H295R ( D ) cell lines. Luciferase reporter assay after transiently transfecting BD140A ( E ) and H295R ( F ) cell lines with wild-type, site A (G644C) and site B (C910G) mutants in the APOBEC3B promoter region.

Article Snippet: Four lentiviral psi-LVRU6P-01, psi-LVRU6P-02, psi-LVRU6P-03 and psi-LVRU6P-04 vectors (catalog # CS-HSH022939-LVRU6P-01 to LVRU6P-04) with U6 promoter expressing shRNAs targeting APOBEC3B and one shRNA scrambled control clone for psi-LVRU6P (catalog # CSHCTR001-LVRU6P) were obtained from GeneCopoeia (Rockville, MD, USA).

Techniques: Binding Assay, Agarose Gel Electrophoresis, SYBR Green Assay, Produced, Chromatin Immunoprecipitation, Negative Control, Positive Control, Biomarker Discovery, Luciferase, Reporter Assay

Journal: Oncotarget

Article Title: GATA3 and APOBEC3B are prognostic markers in adrenocortical carcinoma and APOBEC3B is directly transcriptionally regulated by GATA3

doi: 10.18632/oncotarget.27703

Figure Lengend Snippet: ( A ) APOBEC3B protein expression in H295R cells stably transfected with four different shRNAs targeting APOBEC3B. shRNA 2 and shRNA 4 were selected for in vivo experiments based on the efficiency. ( B ) Tumor growth curve in NOD scid gamma (NSG) mice that received a vector control, APOBEC3B shRNA2, and 4 stably transfected H295R cells flanks on either side. A total of eight mice per each group were used for this study. Three of the control mice died in the first week after injection for unrelated reasons. The error bars represent the standard error mean (SEM). * Indicates p < 0.05. ( C ) The tumor weight of each group at the time of sacrifice (8 weeks after xenograft). ( D ) The isolated tumors from each group at the time of sacrifice.

Article Snippet: Four lentiviral psi-LVRU6P-01, psi-LVRU6P-02, psi-LVRU6P-03 and psi-LVRU6P-04 vectors (catalog # CS-HSH022939-LVRU6P-01 to LVRU6P-04) with U6 promoter expressing shRNAs targeting APOBEC3B and one shRNA scrambled control clone for psi-LVRU6P (catalog # CSHCTR001-LVRU6P) were obtained from GeneCopoeia (Rockville, MD, USA).

Techniques: Expressing, Stable Transfection, Transfection, shRNA, In Vivo, Plasmid Preparation, Control, Injection, Isolation