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mouse monoclonal pax8 abcam (Danaher Inc)

Name: mouse monoclonal pax8 abcam
Brand: Danaher Inc
Rating: 86 (1 reviews)

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Danaher Inc mouse monoclonal pax8 abcam
Mouse Monoclonal Pax8 Abcam, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Expression of PAX8 in clinicopathological characteristics of SCLC.

Journal: Heliyon

Article Title: Prognostic value of PAX8 in small cell lung cancer

doi: 10.1016/j.heliyon.2024.e28251

Figure Lengend Snippet: Expression of PAX8 in clinicopathological characteristics of SCLC.

Article Snippet: The samples were incubated with anti-Mouse PAX8 monoclonal antibody (1:1 dilution; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd; clone number: OTI6H8) or anti-Mouse Ki-67 monoclonal antibody (1:2500 dilution; Beijing Zhongshan Golden Bridge Biotechnology; clone number: UMAB107).

Techniques: Expressing

Immunohistochemical expression of PAX8 in SCLC. (A) Extensive-stage SCLC PAX8 positive case. (B) Limited-stage small cell lung cancer PAX8 positive case. (C) Extensive-stage small cell lung cancer PAX8 negative case. (D) limited-stage small cell lung cancer PAX8 negative case.

Journal: Heliyon

Article Title: Prognostic value of PAX8 in small cell lung cancer

doi: 10.1016/j.heliyon.2024.e28251

Figure Lengend Snippet: Immunohistochemical expression of PAX8 in SCLC. (A) Extensive-stage SCLC PAX8 positive case. (B) Limited-stage small cell lung cancer PAX8 positive case. (C) Extensive-stage small cell lung cancer PAX8 negative case. (D) limited-stage small cell lung cancer PAX8 negative case.

Techniques: Immunohistochemical staining, Expressing

Overexpression of PAX8 in HEK-293T. (A) Real-time PCR analysis of PAX8 in HEK-293T. (B) Immunohistochemical expression of PAX8 in HEK-293T.

Journal: Heliyon

Article Title: Prognostic value of PAX8 in small cell lung cancer

doi: 10.1016/j.heliyon.2024.e28251

Figure Lengend Snippet: Overexpression of PAX8 in HEK-293T. (A) Real-time PCR analysis of PAX8 in HEK-293T. (B) Immunohistochemical expression of PAX8 in HEK-293T.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Expressing

Comparative survival analyses in SCLC patients. (A) Comparison of OS between PAX8 positive and PAX8 negative group. (B) Comparison of OS between Ki-67 high and Ki-67 low group. (C) Comparison of OS between Limited-stage and Extensive-stage patients. (D) Comparison of OS among PAX8 expression and stage status combination groups. The difference in OS between groups was compared by Log-rank test, with death as the outcome.

Journal: Heliyon

Article Title: Prognostic value of PAX8 in small cell lung cancer

doi: 10.1016/j.heliyon.2024.e28251

Figure Lengend Snippet: Comparative survival analyses in SCLC patients. (A) Comparison of OS between PAX8 positive and PAX8 negative group. (B) Comparison of OS between Ki-67 high and Ki-67 low group. (C) Comparison of OS between Limited-stage and Extensive-stage patients. (D) Comparison of OS among PAX8 expression and stage status combination groups. The difference in OS between groups was compared by Log-rank test, with death as the outcome.

Techniques: Comparison, Expressing

The HR value of Ki-67, PAX8 and stage status for overall survival.

Journal: Heliyon

Article Title: Prognostic value of PAX8 in small cell lung cancer

doi: 10.1016/j.heliyon.2024.e28251

Figure Lengend Snippet: The HR value of Ki-67, PAX8 and stage status for overall survival.

Techniques:

a , b Schematic illustration of the experiment and Uniform Manifold Approximation and Projection (UMAP) plot of cells isolated from the proximal part of the testis of P14 Sox17- cKO and controls, showing twenty clusters ( a ) and the distribution of cells from each sample ( b ). Control n = 2, Sox17- cKO n = 2. c UMAP plot showing the expression level of Sox9, Pax8, and Sox17 in the control samples. The right two panels show the expression of each gene in the extracted clusters of the RT (cluster 16) and Sertoli cells (clusters 1, 2, 10, 18). d Dot plots representing the expression levels of marker genes for Sertoli cells ( Amh ) and RT epithelial cells ( Krt8, Pax8 ) together with their common marker genes ( Nr5a1, Gata4, Wt1, Sox9 ) in the clusters expressing Sox9 shown in ( c ) (clusters 1, 2, 10, 16, 18). e A violin plot showing the expression level of Sox17 in each cluster, together with Pecam1 (vascular endothelial cell marker) and Pax8 (RT marker). “ n ” represents the number of biological replicates.

Journal: Nature Communications

Article Title: SOX17-positive rete testis epithelium is required for Sertoli valve formation and normal spermiogenesis in the male mouse

doi: 10.1038/s41467-022-35465-1

Figure Lengend Snippet: a , b Schematic illustration of the experiment and Uniform Manifold Approximation and Projection (UMAP) plot of cells isolated from the proximal part of the testis of P14 Sox17- cKO and controls, showing twenty clusters ( a ) and the distribution of cells from each sample ( b ). Control n = 2, Sox17- cKO n = 2. c UMAP plot showing the expression level of Sox9, Pax8, and Sox17 in the control samples. The right two panels show the expression of each gene in the extracted clusters of the RT (cluster 16) and Sertoli cells (clusters 1, 2, 10, 18). d Dot plots representing the expression levels of marker genes for Sertoli cells ( Amh ) and RT epithelial cells ( Krt8, Pax8 ) together with their common marker genes ( Nr5a1, Gata4, Wt1, Sox9 ) in the clusters expressing Sox9 shown in ( c ) (clusters 1, 2, 10, 16, 18). e A violin plot showing the expression level of Sox17 in each cluster, together with Pecam1 (vascular endothelial cell marker) and Pax8 (RT marker). “ n ” represents the number of biological replicates.

Article Snippet: The sections were incubated overnight at 4 °C with the following primary antibodies [format: host anti-protein (company, catalog number, dilution)]: mouse monoclonal anti-ace-TUB (Sigma, T6793, 1:200), mouse monoclonal anti-αSMA/ACTA2 (Sigma, A5228, 1:500), goat polyclonal anti-AMH (Santa Cruz, sc6886, 1:200), mouse monoclonal anti-CDH1 (BD Transduction lab, 610181, 1:400), goad polyclonal anti-c-KIT (R&D systems, AF1356, 1:200), rabbit monoclonal anti-EPCAM (Abcam, ab32392, 1:100), goat polyclonal anti-GATA4 (Santa Cruz, sc1237, 1:200), mouse monoclonal anti-GATA4 (Santa Cruz, sc25310, 1:100), rabbit polyclonal anti-GFP (MBL, 598, 1:200), goat polyclonal GFRα1 (R&D systems, AF560, 1:100), rabbit polyclonal anti-HSP70 (Abcam, ab79852, 1:1000), rabbit polyclonal anti-KI67 (Abcam, ab15580, 1:400), rabbit monoclonal anti-KRT8 (Abcam, ab53280, 1:200), rabbit polyclonal LAMININ (Abcam, ab11575, 1:200), rabbit monoclonal anti-p-AKT (Cell Signaling, 4060, 1:100), mouse monoclonal anti-PAX8 (Abcam, ab53490, 1:100), rat monoclonal PECAM1 (Invitrogen, 14-0311-82, 1:100), rabbit polyclonal anti-PLZF (Santa Cruz, sc22839, 1:200), mouse monoclonal SCP3 (Santa Cruz, sc74569, 1:500), goat polyclonal anti-SOX17 (R&D systems, AF1924, 1:200), rabbit polyclonal anti-SOX9 (Millipore, AB5535, 1:400), rabbit polyclonal anti-STAR (Cell Signaling, 8449, 1:100), rabbit polyclonal anti-VASA/MVH (Abcam, ab13840, 1:10000).

Techniques: Isolation, Expressing, Marker

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).

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

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.

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.

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

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).

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).

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

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.

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.

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

Journal: Cell Death Discovery

Article Title: Paired box 8 facilitates the c-MYC related cell cycle progress in TP53- mutation uterine corpus endometrial carcinoma through interaction with DDX5

doi: 10.1038/s41420-022-01072-8

Figure Lengend Snippet: A – C PAX8 mRNA levels in normal and cancer tissues of Uterine Corpus Endometrial Carcinoma (UCEC), Uterine Carcinosarcoma (UCS) and Ovarian serous cystadenocarcinoma (OSC) from TNMPLOT online database. D The PAX8 gene expression levels between adjacent normal tissue and paired UCEC tissue (TNMPLOT online database). E The protein level of PAX8 between normal tissues ( n = 33) and primary tumor tissues ( n = 100) from UALCAN online database. Z-values represent standard deviations from the median across samples for the given cancer type. Log2 Spectral count ratio values from CPTAC were first normalized within each sample profile, then normalized across samples. F Kaplan–Meier plotter was used to analysis the effect of PAX8 expression on overall survival in patients with endometrial cancer. HR = 2.05 (1.35–3.11), logran p = 0.00057. G , H qRT-PCR and Western blot were used to verify PAX8 mRNA and protein level in cells: ThE, 12Z, ZQ19, HeLa, Ishikawa, HEC-1B respectively. I Immunohistochemistry was used to detect the expression of PAX8 in endometrial carcinoma. J The relationship between the expression of PAX8 and pathological grade of endometrial carcinoma was analyzed by HistoQuest single-cell analysis (Tissue Gnostics GmbH, Vienna, Austria). * p < 0.05, *** p < 0.001.

Article Snippet: The protein was transferred to the polyvinylidene membranes (PVDF), then primary antibodies immunoblotted with PVDF, primary antibodies including anti-PAX8 rabbit monoclonal antibody (1:1000, Abcam), anti-PAX8 mouse monoclonal antibody (1:2000, Proteintech), anti-DDX5 rabbit polyclonal antibody (1:200, Proteintech), anti-c-MYC mouse polyclonal antibody(1:2000, Proteintech), anti-Lamin B1 rabbit polyclonal antibody (1:2000, Proteintech), anti-HA tag rabbit polyclonal antibody (1:2000, Proteintech), anti-GFP tag rabbit polyclonal antibody (1:2000, Proteintech), anti-Cyclin D1 mouse monoclonal antibody (1:10000, Proteintech), anti-CDK6 mouse monoclonal antibody (1:10000, Proteintech), anti-CDK4 rabbit polyclonal antibody (1:1000, Proteintech), anti-MCM7 rabbit polyclonal antibody (1:2000, Proteintech), anti-GAPDH mouse polyclonal antibody (1:5000, Santa Cruz Biotechnology), anti-β-actin (ACTB) rabbit polyclonal antibody (1:1000, Boster, Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Single-cell Analysis

A The methylation levels of PAX8 (pair3) in HEC-1B, Ishikawa, 12Z, and HeLa cells were detected by bisulfite conversion sequencing. B Schematic diagram of methylation information of all sites in pair3. C Statistical table of methylation information for all sites in pair3. D Promoter methylation level of PAX8 in UCEC was analyzed by TCGA samples. E Promoter methylation level of PAX8 in UCEC based on TP53 muation status was analyzed by TCGA samples. The Beta value indicates level of DNA methylation ranging from 0 (unmethylated) to 1 (fully methylated). F Expression of PAX8 in UCEC based on TP53 muation status was analyzed by TCGA samples. G Protein level of PAX8 in UCEC based on p53-related pathway. H The proportion of PAX8 copy amplification/mRNA upregulation (Altered group) in four molecular subtypes of endometrial cancer. * p < 0.01, ** p < 0.005, *** p < 0.001.

Journal: Cell Death Discovery

Article Title: Paired box 8 facilitates the c-MYC related cell cycle progress in TP53- mutation uterine corpus endometrial carcinoma through interaction with DDX5

doi: 10.1038/s41420-022-01072-8

Figure Lengend Snippet: A The methylation levels of PAX8 (pair3) in HEC-1B, Ishikawa, 12Z, and HeLa cells were detected by bisulfite conversion sequencing. B Schematic diagram of methylation information of all sites in pair3. C Statistical table of methylation information for all sites in pair3. D Promoter methylation level of PAX8 in UCEC was analyzed by TCGA samples. E Promoter methylation level of PAX8 in UCEC based on TP53 muation status was analyzed by TCGA samples. The Beta value indicates level of DNA methylation ranging from 0 (unmethylated) to 1 (fully methylated). F Expression of PAX8 in UCEC based on TP53 muation status was analyzed by TCGA samples. G Protein level of PAX8 in UCEC based on p53-related pathway. H The proportion of PAX8 copy amplification/mRNA upregulation (Altered group) in four molecular subtypes of endometrial cancer. * p < 0.01, ** p < 0.005, *** p < 0.001.

Techniques: Methylation, Sequencing, DNA Methylation Assay, Expressing, Amplification

A Ishikawa cell was transfected with PAX8 siRNA for 24 h, RNA isolated, and RNA-Seq performed. The expression level of PAX8 mRNA after knockdown PAX8 was detected by qPCR. B , C The expression level of PAX8 protein was detected by Western blotting, after knockdown PAX8 in Ishikawa and HEC-b cells. D Volcano plots of differentially expression genes (red means up-regulated genes, green means down-regulated genes). E Categories from the top six families in KEEG pathways analysis of significant gene expression changes under siPAX8. F Differentially expressed genes clustering diagram. The columns represent different samples (three groups on the left are the siNC, and the three groups on the right are siPAX8), the rows represent different genes, and are clustered by lg (TPM + 1) value, red indicates high expression genes, and blue indicates low expression genes. G qPCR was used to verify ribosome, ribosome synthesis, lysosome, and cell cycle-related gene mRNA expression level in Ishikawa cells after knockdown PAX8. H Representative enrichment plots of the ribosome, ribosome synthesis, lysosome, and cell cycle-related gene set which positively or negatively correlate with siPAX8.

Journal: Cell Death Discovery

Article Title: Paired box 8 facilitates the c-MYC related cell cycle progress in TP53- mutation uterine corpus endometrial carcinoma through interaction with DDX5

doi: 10.1038/s41420-022-01072-8

Figure Lengend Snippet: A Ishikawa cell was transfected with PAX8 siRNA for 24 h, RNA isolated, and RNA-Seq performed. The expression level of PAX8 mRNA after knockdown PAX8 was detected by qPCR. B , C The expression level of PAX8 protein was detected by Western blotting, after knockdown PAX8 in Ishikawa and HEC-b cells. D Volcano plots of differentially expression genes (red means up-regulated genes, green means down-regulated genes). E Categories from the top six families in KEEG pathways analysis of significant gene expression changes under siPAX8. F Differentially expressed genes clustering diagram. The columns represent different samples (three groups on the left are the siNC, and the three groups on the right are siPAX8), the rows represent different genes, and are clustered by lg (TPM + 1) value, red indicates high expression genes, and blue indicates low expression genes. G qPCR was used to verify ribosome, ribosome synthesis, lysosome, and cell cycle-related gene mRNA expression level in Ishikawa cells after knockdown PAX8. H Representative enrichment plots of the ribosome, ribosome synthesis, lysosome, and cell cycle-related gene set which positively or negatively correlate with siPAX8.

Techniques: Transfection, Isolation, RNA Sequencing Assay, Expressing, Western Blot

A Representative enrichment plots of the c-MYC-related gene set which negatively correlate with siPAX8 in Ishikawa cell. B PAX8 expression was knocked down by siRNA transfection, cells were collected 48 h after transfection, the expression level of PAX8 and c-MYC proteins in Ishikawa and HEC-1B cells were detected by Western blotting. C After gradient knockdown of PAX8 in Ishikawa cells, the expression level of PAX8 and cell cycle-related proteins were detected by Western blotting. D , E PAX8 was overexpressed by transfected HA-PAX8 plasmid (0ug, 0.5ug, 1ug, 1.5ug, 2ug) in 12Z cells, after 48 h transfection, total RNA and total protein were extracted from cells for qPCR and Western blot analysis, respectively. F Linear regression and correlation of PAX8 versus c-MYC protein level. R value is Pearson’s correlation coefficient. G The mRNA expression level of c-MYC was positively correlated with PAX8 in GEPIA. N.S. not significantly different among the groups by Student’s t-test. * p < 0.01, ** p < 0.005, *** p < 0.001.

Journal: Cell Death Discovery

Article Title: Paired box 8 facilitates the c-MYC related cell cycle progress in TP53- mutation uterine corpus endometrial carcinoma through interaction with DDX5

doi: 10.1038/s41420-022-01072-8

Figure Lengend Snippet: A Representative enrichment plots of the c-MYC-related gene set which negatively correlate with siPAX8 in Ishikawa cell. B PAX8 expression was knocked down by siRNA transfection, cells were collected 48 h after transfection, the expression level of PAX8 and c-MYC proteins in Ishikawa and HEC-1B cells were detected by Western blotting. C After gradient knockdown of PAX8 in Ishikawa cells, the expression level of PAX8 and cell cycle-related proteins were detected by Western blotting. D , E PAX8 was overexpressed by transfected HA-PAX8 plasmid (0ug, 0.5ug, 1ug, 1.5ug, 2ug) in 12Z cells, after 48 h transfection, total RNA and total protein were extracted from cells for qPCR and Western blot analysis, respectively. F Linear regression and correlation of PAX8 versus c-MYC protein level. R value is Pearson’s correlation coefficient. G The mRNA expression level of c-MYC was positively correlated with PAX8 in GEPIA. N.S. not significantly different among the groups by Student’s t-test. * p < 0.01, ** p < 0.005, *** p < 0.001.

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation

$A PAX8-interacting proteins in Ishikawa cell were precipitated by using anti-PAX8 antibody, and identified by LC-MS/MS analyses. The proteins identified only in PAX8 precipitates are listed. B Co-IP analyses of HA-PAX8 and DDX5-GFP interaction in HEK293 cells. C Western analyses of the nuclear and cytoplasmic fractionated samples from 12Z and Ishikawa cells. Lamin B and tubulin were used as the nuclear (N) and cytoplasmic (C) markers, respectively. D Immunofluorescence was used to determine the localization of PAX8 and DDX5 in 12Z and Ishikawa cells. DAPI staining was included to visualize the cell nucleus (Blue). Scale bar = 200 μm in 100x and 100μm in 200x vision. E PAX8 was knocked down in Ishikawa cells by using siRNA, after 48 h transfection, total protein was extracted from cells for Western blot analysis. F Gradient transfection of DDX5 plasmid in 12z cells, 24 h later, RNA was extracted to detect the expression of DDX5, c-MYC, and PAX8, Hprt was used as a housekeeping gene.$

Journal: Cell Death Discovery

Article Title: Paired box 8 facilitates the c-MYC related cell cycle progress in TP53- mutation uterine corpus endometrial carcinoma through interaction with DDX5

doi: 10.1038/s41420-022-01072-8

Figure Lengend Snippet: A PAX8-interacting proteins in Ishikawa cell were precipitated by using anti-PAX8 antibody, and identified by LC-MS/MS analyses. The proteins identified only in PAX8 precipitates are listed. B Co-IP analyses of HA-PAX8 and DDX5-GFP interaction in HEK293 cells. C Western analyses of the nuclear and cytoplasmic fractionated samples from 12Z and Ishikawa cells. Lamin B and tubulin were used as the nuclear (N) and cytoplasmic (C) markers, respectively. D Immunofluorescence was used to determine the localization of PAX8 and DDX5 in 12Z and Ishikawa cells. DAPI staining was included to visualize the cell nucleus (Blue). Scale bar = 200 μm in 100x and 100μm in 200x vision. E PAX8 was knocked down in Ishikawa cells by using siRNA, after 48 h transfection, total protein was extracted from cells for Western blot analysis. F Gradient transfection of DDX5 plasmid in 12z cells, 24 h later, RNA was extracted to detect the expression of DDX5, c-MYC, and PAX8, Hprt was used as a housekeeping gene.

Techniques: Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Expressing

A EdU incorporation assays of HEC-1b cell transiently transfected with siNC or siPAX8 or c-MYC or siPAX8 + c-MYC. B The percentage of EdU positive cells was blindly calculated with counting nine nonoverlaping fields. Values are means ± s.d. C HEC-1B cells were transfected with siRNAs or plasmid to knockdown PAX8 or overexpression c-MYC and DDX5, respectively. The proteins were extracted from cells for Western blotting analysis. D , E HEC-1B cells were transfected with plasmid or siRNA to knockdown PAX8 and overexpression c-MYC and DDX5, after treatment for 48 h. Cells were assayed by flow cytometry. The percentages of the cells in G1, S, and G2 phase were counted and compared. F HEC-1b cells were transfected with siPAX8 or siPAX8 + c-MYC or siPAX8 + DDX5. Representative G1/S transition markers were immunoblotted.

Journal: Cell Death Discovery

Article Title: Paired box 8 facilitates the c-MYC related cell cycle progress in TP53- mutation uterine corpus endometrial carcinoma through interaction with DDX5

doi: 10.1038/s41420-022-01072-8

Figure Lengend Snippet: A EdU incorporation assays of HEC-1b cell transiently transfected with siNC or siPAX8 or c-MYC or siPAX8 + c-MYC. B The percentage of EdU positive cells was blindly calculated with counting nine nonoverlaping fields. Values are means ± s.d. C HEC-1B cells were transfected with siRNAs or plasmid to knockdown PAX8 or overexpression c-MYC and DDX5, respectively. The proteins were extracted from cells for Western blotting analysis. D , E HEC-1B cells were transfected with plasmid or siRNA to knockdown PAX8 and overexpression c-MYC and DDX5, after treatment for 48 h. Cells were assayed by flow cytometry. The percentages of the cells in G1, S, and G2 phase were counted and compared. F HEC-1b cells were transfected with siPAX8 or siPAX8 + c-MYC or siPAX8 + DDX5. Representative G1/S transition markers were immunoblotted.

Techniques: Transfection, Plasmid Preparation, Over Expression, Western Blot, Flow Cytometry

In TP53 wild-type cells, a small amount of PAX8 is insufficient to co-regulate the transcriptional level of c-MYC with DDX5, while in TP53-mutated cells, promoter hypomethylation leads to high expression of PAX8, which in turn cooperates with DDX5, possibly by unraveling the Myc4G structure leading to increased c-MYC expression and promotes cell cycle progression.

Journal: Cell Death Discovery

Article Title: Paired box 8 facilitates the c-MYC related cell cycle progress in TP53- mutation uterine corpus endometrial carcinoma through interaction with DDX5

doi: 10.1038/s41420-022-01072-8

Figure Lengend Snippet: In TP53 wild-type cells, a small amount of PAX8 is insufficient to co-regulate the transcriptional level of c-MYC with DDX5, while in TP53-mutated cells, promoter hypomethylation leads to high expression of PAX8, which in turn cooperates with DDX5, possibly by unraveling the Myc4G structure leading to increased c-MYC expression and promotes cell cycle progression.

Techniques: Expressing

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