Journal: Oncotarget

Article Title: Loss of calponin h1 confers anoikis resistance and tumor progression in the development of high-grade serous carcinoma originating from the fallopian tube epithelium

doi: 10.18632/oncotarget.18024

Figure Lengend Snippet: ( A ) Human fimbria epithelial cells (FTE) were primarily cultured, transduced with pLenti-E6/E7 and pLenti-hTERT. The immortalized cell line FE25 was derived. By additional transduction with pLenti-Ras V12 oncogene, FE-RAS cells were derived. Western blot results showed the expression pattern of RAS, PAX8, p53, HPV E6 and E7 in FE25 and FE-RAS cells. ( B ) Immunohistochemistry of xenotumors from FE-RAS cells subcutaneous (upper panel) and intraperitoneal (lower panel) injections into NSG mice. Scale bars: 50 μm.

Article Snippet: The primary antibodies included rabbit anti-p53 monoclonal antibody (#2527, 1:1000; Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-RAS (1:1000; Abcam), mouse monoclonal anti-PAX8 (clone PAX8R1, 1:1000; Abcam), mouse monoclonal anti-HPV16+HPV18 E6 (C1P5, 1:1000; Abcam), mouse monoclonal anti-HPV16 E7 (289–17013, 1:1000; Abcam), rabbit anti-CNN1 (EP798Y, 1:2500; Abcam), mouse anti-Actin (MM2/193, 1:10000; Santa Cruz Biotechnology), rabbit anti-integrin alpha 2/CD49b (1:1000; Bioss, Woburn, MA, USA), and rabbit anti-integrin beta 1 (1:1000; Bioss).

Techniques: Cell Culture, Transduction, Derivative Assay, Western Blot, Expressing, Immunohistochemistry

Journal: BMC Molecular Biology

Article Title: Pax8 modulates the expression of Wnt4 that is necessary for the maintenance of the epithelial phenotype of thyroid cells

doi: 10.1186/1471-2199-15-21

Figure Lengend Snippet: Wnt4 is localized in the cytoplasmic compartment of FRTL-5 cells and its expression is modulated by TSH. A) Wnt4 expression is modulated by TSH in FRTL-5 thyroid cells. FRTL-5 cells were cultured in regular medium or in starvation medium for 4 days (0.2% CS) and treated with 1 mU/ml of TSH for 24 h, and analyzed by immunofluorescence to detect Pax8 and Wnt4 proteins with specific antibodies (see Methods). B) TSH stimulation of Wnt4 expression is mediated by Pax8. FRTL-5 cells were cultured in regular medium and transfected with 100 nM of siPax8 or siCtrl-. Seven hours later, cells were starved in 0.2% CS medium for 24 h and treated with 1 mU/ml of TSH for 3 h and analyzed by qRT-PCR to measure the expression levels of Pax8 (black bars) and Wnt4 (gray bars) mRNAs. Data are from three independent experiments, each performed in duplicate and are expressed as the mean ± SD (P < 0,1).

Article Snippet: Briefly, the cells were subsequently blocked with washing buffer containing 0.5% BSA for 1 h and treated with rabbit policlonal anti-WNT4 (Wnt4 antibody, Novus) O/N and then with Alexa Fluor ® 594-conjugated secondary antibody (Invitrogen) for 30 min, followed by treatment with mouse monoclonal anti-PAX8 (BIOCARE MEDICAL) for 1 h and finally by an Alexa Fluor ® 488-conjugated secondary antibody (Invitrogen).

Techniques: Expressing, Cell Culture, Immunofluorescence, Transfection, Quantitative RT-PCR

Journal: BMC Molecular Biology

Article Title: Pax8 modulates the expression of Wnt4 that is necessary for the maintenance of the epithelial phenotype of thyroid cells

doi: 10.1186/1471-2199-15-21

Figure Lengend Snippet: Pax8 activates transcription from the Wnt4 5′-flanking region. A) Transcriptional activity of Wnt4 5′-flanking genomic region. The deletion reporter constructs were transfected into FRTL-5 and HeLa cells and the luciferase activity was determined. All the constructs have the highest activity in thyroid cells and only the 190Wnt4LUC construct shows a strongly reduced activity. Data are expressed as the mean ± SD (P < 0,0001); B) Pax8 activates transcription from the Wnt4 promoter region. HeLa cells were transiently transfected with 300Wnt4LUC and 190Wnt4LUC reporter constructs alone and in combination with increasing concentration (100 and 200 ng) of the expression vector encoding Pax8 (CMV5-Pax8). The transcriptional activity was determined 48 h after transfection as the firefly over renilla luciferase activity. Data are expressed as fold induction over the transcription obtained with 300Wnt4LUC and 190Wnt4LUC, whose values were set at 1. Data are from three independent experiments, each performed in duplicate and are expressed as the mean ± SD (P < 0,005); C) Pax8 binds the Wnt4 promoter region in vivo. Chromatin extracted from cross-linked FRTL-5 cells was immunoprecipitated with an unrelated antibody or for Pax8. The immunoprecipitates were analyzed by PCR with oligonucleotides able to amplify the region between -300 bp and -190 bp of the 5′flanking region of the Wnt4 gene.

Article Snippet: Briefly, the cells were subsequently blocked with washing buffer containing 0.5% BSA for 1 h and treated with rabbit policlonal anti-WNT4 (Wnt4 antibody, Novus) O/N and then with Alexa Fluor ® 594-conjugated secondary antibody (Invitrogen) for 30 min, followed by treatment with mouse monoclonal anti-PAX8 (BIOCARE MEDICAL) for 1 h and finally by an Alexa Fluor ® 488-conjugated secondary antibody (Invitrogen).

Techniques: Activity Assay, Construct, Transfection, Luciferase, Concentration Assay, Expressing, Plasmid Preparation, In Vivo, Immunoprecipitation

Journal: BMC Molecular Biology

Article Title: Pax8 modulates the expression of Wnt4 that is necessary for the maintenance of the epithelial phenotype of thyroid cells

doi: 10.1186/1471-2199-15-21

Figure Lengend Snippet: Wnt4 5′-flanking region contains a binding site for Pax8. A) Graphic output of the sequence analysis showing the conservation in different species of the core consensus sequences (in bold) of Pax8 binding sites. The sequence alignment was obtained using whole genome comparative analysis of the VISTA browser; B) Electromobility shift assays for Pax8 binding to putative recognition motifs in the Wnt4 promoter. Left panel, 32 P-labeled oligo probes A and B were challenged with total protein extracts prepared from FRTL-5 cells (lane 2). Protein extracts of HeLa (lane 3) and Pax8-transfected HeLa cells (lane 4) were used as negative and positive control, respectively. Middle panel, the specificity of the complex observed with the FRTL-5 extract and probe A was tested by competition analysis, using increasing amount (from 25 to 100-fold molar excess) of unlabeled wild-type oligo A or mutated in the core sequence. Right panel, the FRTL-5 protein extract was incubated with the antibody against Pax8 or tubulin (as negative control), in a supershift EMSA with labeled probe A; C) Pax8 binding site A is necessary for the transcriptional activity of Wnt4 promoter. The deletion constructs 300Wnt4LUC and 260Wnt4LUC were transfected into FRTL-5 thyroid cells and the luciferase activity was determined. Data are from three independent experiments, each performed in duplicate and are expressed as the mean ± SD (P < 0,05).

Article Snippet: Briefly, the cells were subsequently blocked with washing buffer containing 0.5% BSA for 1 h and treated with rabbit policlonal anti-WNT4 (Wnt4 antibody, Novus) O/N and then with Alexa Fluor ® 594-conjugated secondary antibody (Invitrogen) for 30 min, followed by treatment with mouse monoclonal anti-PAX8 (BIOCARE MEDICAL) for 1 h and finally by an Alexa Fluor ® 488-conjugated secondary antibody (Invitrogen).

Techniques: Binding Assay, Sequencing, Labeling, Transfection, Positive Control, Incubation, Negative Control, Activity Assay, Construct, Luciferase

Journal: BMC Molecular Biology

Article Title: Pax8 modulates the expression of Wnt4 that is necessary for the maintenance of the epithelial phenotype of thyroid cells

doi: 10.1186/1471-2199-15-21

Figure Lengend Snippet: Pax8 and Wnt4 expression in human thyroid cancer cell lines. Wnt4 and Pax8 expression was measured by qRT-PCR in four human thyroid cancer cell lines: WRO from follicular thyroid cancer, Cal62 from anaplastic thyroid carcinoma, FB2 and BCPAP from papillary thyroid carcinoma. RNA from six non-pathological thyroids (N.T.) was used as control. Quantitative real-time PCR was carried out in triplicate as described in Materials and Methods. Glyceraldehyde 3-phosphate dehydrogenase was used as reference gene; results are reported as 2 -∆Ct . Data are expressed as the mean ± SD (P < 0,05). The bottom panel shows the total protein extracts of FRTL-5 cells (used as normal thyroid) and human thyroid cancer cells WRO, Cal62, FB2 and BCPAP separated on SDS-PAGE and subjected to Western blot analysis with specific antibodies as indicated in the panel. The hybridization with GAPDH assessed the protein uniform loading integrity. Fold represents the quantification of protein levels normalized with GAPDH.

Article Snippet: Briefly, the cells were subsequently blocked with washing buffer containing 0.5% BSA for 1 h and treated with rabbit policlonal anti-WNT4 (Wnt4 antibody, Novus) O/N and then with Alexa Fluor ® 594-conjugated secondary antibody (Invitrogen) for 30 min, followed by treatment with mouse monoclonal anti-PAX8 (BIOCARE MEDICAL) for 1 h and finally by an Alexa Fluor ® 488-conjugated secondary antibody (Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SDS Page, Western Blot, Hybridization

Journal: Oncotarget

Article Title: Somatic mutations and increased lymphangiogenesis observed in a rare case of intramucosal gastric carcinoma with lymph node metastasis

doi: 10.18632/oncotarget.24289

Figure Lengend Snippet: The list of target genes examined in panel sequencing

Article Snippet: Primary antibodies used were the mouse monoclonal anti-PAX8 antibody (1:200 dilution) (Santa Cruz Biotechnology, Dallas, TX, USA), the mouse monoclonal anti-V5 antibody (1:5000 dilution) (Thermo Fisher Scientific), and the mouse monoclonal anti-β-actin antibody (1:1000 dilution) (Sigma).

Techniques:

Journal: Oncotarget

Article Title: Somatic mutations and increased lymphangiogenesis observed in a rare case of intramucosal gastric carcinoma with lymph node metastasis

doi: 10.18632/oncotarget.24289

Figure Lengend Snippet: Annotations for somatic mutations of NBN and PAX8

Article Snippet: Primary antibodies used were the mouse monoclonal anti-PAX8 antibody (1:200 dilution) (Santa Cruz Biotechnology, Dallas, TX, USA), the mouse monoclonal anti-V5 antibody (1:5000 dilution) (Thermo Fisher Scientific), and the mouse monoclonal anti-β-actin antibody (1:1000 dilution) (Sigma).

Techniques: Mutagenesis

Journal: Oncotarget

Article Title: Somatic mutations and increased lymphangiogenesis observed in a rare case of intramucosal gastric carcinoma with lymph node metastasis

doi: 10.18632/oncotarget.24289

Figure Lengend Snippet: (A) Validation by Sanger sequencing showed single nucleotide substitution in NBN and PAX8 in the lymph node metastasis (arrow) but not in the primary tumor. (B) Functional domains (colored boxes) and mutated residues (arrows) in nibrin (NBN) and paired box 8 (PAX8).

Article Snippet: Primary antibodies used were the mouse monoclonal anti-PAX8 antibody (1:200 dilution) (Santa Cruz Biotechnology, Dallas, TX, USA), the mouse monoclonal anti-V5 antibody (1:5000 dilution) (Thermo Fisher Scientific), and the mouse monoclonal anti-β-actin antibody (1:1000 dilution) (Sigma).

Techniques: Sequencing, Functional Assay

Journal: Oncotarget

Article Title: Somatic mutations and increased lymphangiogenesis observed in a rare case of intramucosal gastric carcinoma with lymph node metastasis

doi: 10.18632/oncotarget.24289

Figure Lengend Snippet: (A) Immunoblot of 293T cells transfected with the PAX8 R49H -V5-His (PAX8 R49H), PAX8 wild-type -V5-His (PAX8 WT), and pcDNA3.1/V5-His vector (Empty vector) probed with antibodies of anti-PAX8, anti-V5, and anti-beta actin. (B) The relative expression of E2F1 in each transfected 293T cells measured by the quantitative real-time PCR assay. The expression of E2F1 was normalized to the expression of GAPDH and analyzed by means of the 2 −ΔΔCT method. An asterisk indicated P < 0.001.

Article Snippet: Primary antibodies used were the mouse monoclonal anti-PAX8 antibody (1:200 dilution) (Santa Cruz Biotechnology, Dallas, TX, USA), the mouse monoclonal anti-V5 antibody (1:5000 dilution) (Thermo Fisher Scientific), and the mouse monoclonal anti-β-actin antibody (1:1000 dilution) (Sigma).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction