Journal: Clinics

Article Title: Isolated familial somatotropinoma: 11q13-loh and gene/protein expression analysis suggests a possible involvement of aip also in non-pituitary tumorigenesis

doi: 10.1590/S1807-59322010000400010

Figure Lengend Snippet: Genealogy: identification of a functional AIP mutation that may disrupt the cAMP signaling pathway. A - The index patient developed acromegaly, adrenocortical carcinoma and B-cell lymphoma. One of her daughters had acromegaly due to a large and invasive somatotropinoma. B - The known AIP functional mutation c.241C>T (R81X) was identified in genomic DNA samples from both patients (mother and daughter) with acromegaly but not in the three unaffected family members.

Article Snippet: Quantitative PCR (qPCR) was performed with an ABI Prism 7700 Sequence Detector using the TaqMan Gene Expression Assays (Hs00610222_m1 for AIP and 43263 for β-actin) following the manufacturer’s instructions (Applied Biosystems).

Techniques: Functional Assay, Mutagenesis

Journal: Clinics

Article Title: Isolated familial somatotropinoma: 11q13-loh and gene/protein expression analysis suggests a possible involvement of aip also in non-pituitary tumorigenesis

doi: 10.1590/S1807-59322010000400010

Figure Lengend Snippet: Loss of AIP in the familial somatotropinoma. A - The MRI of the index patient’s daughter revealed a large and invasive pituitary adenoma (so-matotropinoma) that was resistant to treatment with a somatostatin analog. She inherited the heterozygous R81X AIP germline mutation from the index case. B - The sequence analysis of AIP in tumoral DNA samples revealed that only the R81X-mutated allele (t) was present in the somatotropinoma, indicating somatic loss and inactivation of wild-type AIP . C - The immunohistochemical analysis showed AIP protein expression in the normal pituitary and a GH-secreting pituitary adenoma of a patient with sporadic acromegaly without AIP mutation. The somatotropinoma of the patient harboring the R81X germline mutation and the somatic loss of the gene presented low AIP protein expression.

Article Snippet: Quantitative PCR (qPCR) was performed with an ABI Prism 7700 Sequence Detector using the TaqMan Gene Expression Assays (Hs00610222_m1 for AIP and 43263 for β-actin) following the manufacturer’s instructions (Applied Biosystems).

Techniques: Mutagenesis, Sequencing, Immunohistochemical staining, Expressing

Journal: Clinics

Article Title: Isolated familial somatotropinoma: 11q13-loh and gene/protein expression analysis suggests a possible involvement of aip also in non-pituitary tumorigenesis

doi: 10.1590/S1807-59322010000400010

Figure Lengend Snippet: Loss of AIP in the adrenocortical tumor. A - An abdominal imaging scan of the index case when she presented with virilization and high serum levels of adrenal steroids. The scan revealed a large heterogeneous mass (9.0 cm x 8.9 cm) in the right adrenal gland, which was surgically excised and histopathologically classified as adrenocortical carcinoma. B - Sequence and microsatellite (D11S1258 AIP -flanking marker) analyses of DNA samples from the tumor revealed loss of the wild-type AIP allele. C - The 2 -δ δ CT method was used to compare AIP mRNA expression in pooled normal pituitary glands (Ct AIP=25.66, Ct β-actin=24.24), pooled normal adrenal glands (Ct AIP=25.42; Ct β-actin=22.49) and the adrenocortical carcinoma from the index case, in whom the AIP wild-type allele was lost (Ct AIP=31.53, Ct β-actin=27.28). The mean Ct value of the normal adrenal pool was used as a reference (1.0), by comparison, there was decreased AIP expression in the adrenocortical carcinoma (0.48). D - Immunohistochemistry using the AIP antibody showed low/ moderate staining in a normal adrenal gland obtained from autopsy. A complete loss of AIP immunoreactivity was observed in the adrenocortical carcinoma from the index patient, which stained positive for Melan A and 35betaH11, which were used as positive controls.

Article Snippet: Quantitative PCR (qPCR) was performed with an ABI Prism 7700 Sequence Detector using the TaqMan Gene Expression Assays (Hs00610222_m1 for AIP and 43263 for β-actin) following the manufacturer’s instructions (Applied Biosystems).

Techniques: Imaging, Sequencing, Marker, Expressing, Comparison, Immunohistochemistry, Staining

Journal: Clinics

Article Title: Isolated familial somatotropinoma: 11q13-loh and gene/protein expression analysis suggests a possible involvement of aip also in non-pituitary tumorigenesis

doi: 10.1590/S1807-59322010000400010

Figure Lengend Snippet: Maintenance of heterozygosity and positive AIP immunostaining in the B-cell non-Hodgkin lymphoma of the R81X AIP mutated IFS patient.

Article Snippet: Quantitative PCR (qPCR) was performed with an ABI Prism 7700 Sequence Detector using the TaqMan Gene Expression Assays (Hs00610222_m1 for AIP and 43263 for β-actin) following the manufacturer’s instructions (Applied Biosystems).

Techniques: Immunostaining

Journal: Cancer Cell International

Article Title: ARNT is a potential direct HIF-1 target gene in human Hep3B hepatocellular carcinoma cells

doi: 10.1186/s12935-017-0446-2

Figure Lengend Snippet: Materials

Article Snippet: Briefly, mRNA expression was determined using TaqMan ® Gene Expression Assays (ARNT #Hs01121918_m1, HIF1A #Hs00936368_m1, VEGFA #Hs00900055_m1, Applied Biostems) and normalised to Beta-2-microglobulin (B2 M) mRNA (#Hs00187842_m1, Applied Biosystems).

Techniques: Transduction, Control

Journal: Cancer Cell International

Article Title: ARNT is a potential direct HIF-1 target gene in human Hep3B hepatocellular carcinoma cells

doi: 10.1186/s12935-017-0446-2

Figure Lengend Snippet: Recruitment of HIF-1α and ARNT to the ARNT gene promoter. a Functional elements within the ARNT promoter sequence upstream to −1200 bp from the transcription start site (bp 1). HBS HIF binding site, HAS HIF ancillary sequence, TATA TATA box. Investigated partial sequences are indicated (Region 1, Region 2, Region 3). b Chromatin immunoprecipitation (ChIP) assays. Values are presented as mean ± SEM of n = 3 independent experiments. IgG normal rabbit IgG, H3 histone H3

Article Snippet: Briefly, mRNA expression was determined using TaqMan ® Gene Expression Assays (ARNT #Hs01121918_m1, HIF1A #Hs00936368_m1, VEGFA #Hs00900055_m1, Applied Biostems) and normalised to Beta-2-microglobulin (B2 M) mRNA (#Hs00187842_m1, Applied Biosystems).

Techniques: Functional Assay, Sequencing, Binding Assay, Chromatin Immunoprecipitation

Journal: Cancer Cell International

Article Title: ARNT is a potential direct HIF-1 target gene in human Hep3B hepatocellular carcinoma cells

doi: 10.1186/s12935-017-0446-2

Figure Lengend Snippet: CRISPR/Cas9 genome editing of the ARNT gene promoter. a Location of CRISPR/Cas9 target sequences ( light grey ) within Region 3. HBS HIF binding site, HAS HIF ancillary sequence, PAM protospacer-adjacent motif. b Cleavage detection assay of untransfected (UT), CRISPR/Cas9-Target 1 (T1) or CRISPR/Cas9-Target 2 (T2) transfected Hep3B cells. Fragment sizes are given in bp. The arrow indicates the re-hybridised PCR product. Representative result of n = 3 independent experiments. c. p. cleavage products

Article Snippet: Briefly, mRNA expression was determined using TaqMan ® Gene Expression Assays (ARNT #Hs01121918_m1, HIF1A #Hs00936368_m1, VEGFA #Hs00900055_m1, Applied Biostems) and normalised to Beta-2-microglobulin (B2 M) mRNA (#Hs00187842_m1, Applied Biosystems).

Techniques: CRISPR, Binding Assay, Sequencing, Detection Assay, Transfection

Journal: Cancer Cell International

Article Title: ARNT is a potential direct HIF-1 target gene in human Hep3B hepatocellular carcinoma cells

doi: 10.1186/s12935-017-0446-2

Figure Lengend Snippet: Western blot analysis of CRISPR/Cas9 genome edited Hep3B cells. a Representative Western blot of n = 4 independent experiments. Untransfected (UT), CRISPR/Cas9-Target 1 (T1) or CRISPR/Cas9-Target 2 (T2) transfected Hep3B cells were cultured under normoxic (N) conditions or exposed to hypoxia (H) for 8 h. Protein masses are indicated in kDa. b Densitometry of ARNT protein level (corresponding to a ) normalised to Lamin A/C. Values are presented as mean ± SEM of n = 4 independent experiments

Article Snippet: Briefly, mRNA expression was determined using TaqMan ® Gene Expression Assays (ARNT #Hs01121918_m1, HIF1A #Hs00936368_m1, VEGFA #Hs00900055_m1, Applied Biostems) and normalised to Beta-2-microglobulin (B2 M) mRNA (#Hs00187842_m1, Applied Biosystems).

Techniques: Western Blot, CRISPR, Transfection, Cell Culture

Journal: Cancer Cell International

Article Title: ARNT is a potential direct HIF-1 target gene in human Hep3B hepatocellular carcinoma cells

doi: 10.1186/s12935-017-0446-2

Figure Lengend Snippet: qRT-PCR analysis of CRISPR/Cas9 genome-edited Hep3B cells cultured in Lumox ® gas permeable petri-dishes. a ARNT mRNA- and b HIF1A mRNA expression were measured using TaqMan ® Gene Expression Assays under normoxic and hypoxic (3% O 2 , 5 h) conditions. Values are presented as mean ± SEM of n = 3 independent experiments. N normoxia, H hypoxia

Article Snippet: Briefly, mRNA expression was determined using TaqMan ® Gene Expression Assays (ARNT #Hs01121918_m1, HIF1A #Hs00936368_m1, VEGFA #Hs00900055_m1, Applied Biostems) and normalised to Beta-2-microglobulin (B2 M) mRNA (#Hs00187842_m1, Applied Biosystems).

Techniques: Quantitative RT-PCR, CRISPR, Cell Culture, Expressing, Gene Expression

Journal: Cancer Cell International

Article Title: ARNT is a potential direct HIF-1 target gene in human Hep3B hepatocellular carcinoma cells

doi: 10.1186/s12935-017-0446-2

Figure Lengend Snippet: Proposed mechanism of hypoxia-inducible ARNT expression in Hep3B cells

Article Snippet: Briefly, mRNA expression was determined using TaqMan ® Gene Expression Assays (ARNT #Hs01121918_m1, HIF1A #Hs00936368_m1, VEGFA #Hs00900055_m1, Applied Biostems) and normalised to Beta-2-microglobulin (B2 M) mRNA (#Hs00187842_m1, Applied Biosystems).

Techniques: Expressing