Journal: PloS one

Article Title: Comparative functional genomics analysis of NNK tobacco-carcinogen induced lung adenocarcinoma development in Gprc5a-knockout mice.

doi: 10.1371/journal.pone.0011847

Figure Lengend Snippet: Figure 7. Elevated expression of Ube2c, Mcm2, Fen1 and cyclin D1 proteins in Gprc5a-knockout adenocarcinomas and normal lung tissue and cells. A. Representative photomicrographs depicting increased expression of the protein products of Mcm2, Ube2c, Fen1 and cyclin D1 in NNK-induced adenocarcinomas and normal lung histological tissue specimens obtained from NNK-exposed Gprc5a-knockout mice. All four proteins exhibited mainly nuclear localization of expression. B. The number of cells exhibiting positive reactivity (nuclear staining) was counted in up to three separate microscopic fields per tumor or normal lung in five different NNK-exposed Gprc5a2/2 mice. P-values were obtained by the students two-sample t-test (*, p,0.001). C. Western blotting analysis of the four antigens in three different culture dishes of each of the Gprc5a2/2 MDA-F471 adenocarcinoma and normal lung cells. Approximately 106 cells per 10 cm cell culture dish were harvested to prepare total protein extracts. Samples of these extracts (20 mg protein) were analyzed by western blotting using primary antibodies against the indicated proteins. Membrane blots were also analyzed with antibodies against b-actin to compare protein loading in the different lanes. doi:10.1371/journal.pone.0011847.g007

Article Snippet: Other sections were mounted on poly-L-lysine-coated slides and used for immunohistochemical analysis with antibodies against mouse ubiquitin-conjugating enzyme E2C (Ube2c) (Boston Biochem, Cambridge, MA), flap-like endonuclease 1 (Fen1) (Novus Biologicals, Litteleton, CO), minichromosome maintenance 2 (Mcm2) (Cell Signaling Technology, Danvers, MA), and cyclin D1 (Ccnd1) (Cell Marque Rocklin CA).

Techniques: Expressing, Knock-Out, Staining, Western Blot, Cell Culture, Membrane

Journal: Nucleic Acids Research

Article Title: Short DNA/RNA heteroduplex oligonucleotide interacting proteins are key regulators of target gene silencing

doi: 10.1093/nar/gkab258

Figure Lengend Snippet: Toc-HDO-dependent gene silencing of the identified proteins. ( A ) Hepa1–6 cells were used to generate subcell lines stably expressing shRNA against no target (ctrl), luciferase , ANXA5 , CA8 , RNase H1 , APEX1 and FEN1 . Immunoblots performed with the extracts from shRNA knocked down Hepa1–6 cells. ( B ) Toc-Malat1 dose-dependent gene expression regulation analyzed by RT-qPCR ( n = 3). ( C and D ) Quantitative analysis of the substrates uptake of (C) Toc-Malat1 effect ( n = 4), (D) ASO effect ( n = 4). ( E – H ) Quantitative analysis of the (E) Toc-Malat1 effect with 25 nM ( n = 3, light gray), 50 nM ( n = 5, gray), and 100 nM ( n = 4, dark gray), (F) 100 nM of Toc-DMPK effect ( n = 4), (G) 100 nM of Toc-ApoB effect ( n = 5) or (H) 500 nM of ASO effect against Malat1 ( n = 4).

Article Snippet: Antibodies against ACTIN, ANXA2, ANXA5, APEX1, CA8, FEN1, RNase H1, and TPM3 were obtained from Abcam (APEX1 #ab137708 and FEN1 #ab153825 for immunoblots), GeneTex (APEX1 #GTX110558 for immunoprecipitation), ProteinTech (ANXA2 #11256–1-AP and CA8 #12391-1-AP for immunoblots and immunoprecipitation, ANXA5 #11060-1-AP and RNase H1 #15606-1-AP for immunoblots, and TPM3 #10737–1-AP for immunoprecipitation), Cell Signaling Technology (normal Rabbit IgG #3900 (DAE1) for immunoprecipitaion), and WAKO (ACTIN #017-24573 for immunoblot).

Techniques: Stable Transfection, Expressing, shRNA, Luciferase, Western Blot, Gene Expression, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Short DNA/RNA heteroduplex oligonucleotide interacting proteins are key regulators of target gene silencing

doi: 10.1093/nar/gkab258

Figure Lengend Snippet: In vitro binding assays with purified recombinant proteins. ( A ) CBB stained polyacrylamide gel depicting purified recombinant ANXA5 and CA8 (left panel) and immunoblotting of recombinant CA8 proteins with either an anti-CA8 antibody or anti-His antibody (right panel). ( B and C ) Binding assays of recombinant ANXA5 (0, 250, 500 and 1000 nM) and CA8 (0, 500, 1000 and 2000 nM) with (B) Toc-Malat1 and (C) Toc-DMPK. ( D ) CBB stained polyacrylamide gel depicting purified recombinant APEX1 and FEN1. ( E and F ) In vitro binding assays of Toc-HDO substrates incubated with recombinant APEX1 and FEN1 in (E) N buffer (APEX1: 50, 100 and 150 nM; FEN1: 50, 100 and 150 nM), (F) M buffer with APEX1, FEN1 and RNase H (APEX1: 50, 100 and 150 nM; FEN1: 50, 100 and 150 nM; RNase H: 5, 10 and 20 mU). ( G ) Complete binding of Toc-HDO substrates by high dose of APEX1 and FEN1 in M buffer (APEX1: 100, 200 and 400 nM; FEN1: 100, 200 and 400 nM; RNase H: 1, 2.5, 5 mU). ( H ) Cation-dependent binding pattern of APEX1 (APEX1: 2.7, 27 and 270 nM). ( I ) Substrate length-dependent binding assays with ANXA5, CA8, APEX1 and FEN1 (1000, 1000, 150, 150 nM, respectively). ( J ) In vitro binding assays with or without Toc 13-mer substrates with ANXA5, CA8, APEX1 and FEN1 (1000, 1000, 150, 150 nM, respectively).

Article Snippet: Antibodies against ACTIN, ANXA2, ANXA5, APEX1, CA8, FEN1, RNase H1, and TPM3 were obtained from Abcam (APEX1 #ab137708 and FEN1 #ab153825 for immunoblots), GeneTex (APEX1 #GTX110558 for immunoprecipitation), ProteinTech (ANXA2 #11256–1-AP and CA8 #12391-1-AP for immunoblots and immunoprecipitation, ANXA5 #11060-1-AP and RNase H1 #15606-1-AP for immunoblots, and TPM3 #10737–1-AP for immunoprecipitation), Cell Signaling Technology (normal Rabbit IgG #3900 (DAE1) for immunoprecipitaion), and WAKO (ACTIN #017-24573 for immunoblot).

Techniques: In Vitro, Binding Assay, Purification, Recombinant, Staining, Western Blot, Incubation

Journal: Nucleic Acids Research

Article Title: Short DNA/RNA heteroduplex oligonucleotide interacting proteins are key regulators of target gene silencing

doi: 10.1093/nar/gkab258

Figure Lengend Snippet: Characterization of identified proteins. ( A ) Fluorescence polarization assays of Toc-HDO substrates incubated with recombinant ANXA5, CA8, APEX1, and FEN1 proteins (10 −9 to 10 −5.5 M). ( B and C ) In vitro binding assays with ANXA5, CA8, APEX1, and FEN1 (1000, 1000, 150 and 150 nM, respectively) and Toc-HDO substrates with (B) PS ASO backbone and (C) PO ASO backbones. ( D ) Pull-down experiments using Toc-biotin–HDO and biotin–ASO of Malat1.

Article Snippet: Antibodies against ACTIN, ANXA2, ANXA5, APEX1, CA8, FEN1, RNase H1, and TPM3 were obtained from Abcam (APEX1 #ab137708 and FEN1 #ab153825 for immunoblots), GeneTex (APEX1 #GTX110558 for immunoprecipitation), ProteinTech (ANXA2 #11256–1-AP and CA8 #12391-1-AP for immunoblots and immunoprecipitation, ANXA5 #11060-1-AP and RNase H1 #15606-1-AP for immunoblots, and TPM3 #10737–1-AP for immunoprecipitation), Cell Signaling Technology (normal Rabbit IgG #3900 (DAE1) for immunoprecipitaion), and WAKO (ACTIN #017-24573 for immunoblot).

Techniques: Fluorescence, Incubation, Recombinant, In Vitro, Binding Assay

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Selection and validation of FEN1 and ENDOU as candidate biomarkers. (A) Forest plot illustrating differential expression of FEN1 across studies integrated using MetaVolcanoR. (B) FEN1 transcript expression in TCGA-CESC cohort. (C) Forest plot illustrating differential expression of ENDOU across studies integrated using MetaVolcanoR. (D) Validation of ENDOU expression in TCGA-CESC cohort. For each box plot and Kruskal-Wallis test, the central line represents the median; the outer black lines on the box represent the upper and lower quartiles; dots represent outliers; the black lines are the whiskers, which extend from the interquartile ranges to the maximum values that are not classed as outliers. FEN1, flap structure-specific endonuclease 1;ENDOU, poly (U)-specific endoribonuclease; TCGA, The Cancer Genome Atlas; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; SCC, squamous cell carcinoma; ADC, adenocarcinoma; FC, fold change; CI, confidence interval.

Article Snippet: Antigen retrieval was performed with Tris-EDTA buffer pH 7.8 (Cell Conditioner #1; Ventana Medical System, Inc.) at 95°C for 44 min followed by blocking of endogenous peroxides and proteins with inhibitor CM (Ventana Medical System, Inc.; cat. no. 760-159) at 37°C for 4 min. IHC staining was performed as a fully automated assay in the BenchMark ULTRA automated slide stainer (Ventana Medical System, Inc.) using rabbit polyclonal antibody against FEN1 (1 ng/ml; Atlas antibodies cat. no. HPA006748; http://www.atlasantibodies.com ), ENDOU (2 ng/ml; Atlas antibodies, cat. no. HPA012388; http://www.atlasantibodies.com ), p16 (p16 CINtec ® Histology kit; Roche Diagnostics GmbH; cat no. 06695256001; http://diagnostics.roche.com ) and UltraView DAB IHC Detection kit (cat. no. 760-500; Ventana Medical System, Inc.).

Techniques: Selection, Biomarker Discovery, Quantitative Proteomics, Expressing

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Expression of candidate biomarkers associated with HPV status and genotype. (A) Clustering mRNA expression of the candidate biomarkers based on HPV infection status. (B) Clustering mRNA expression of the candidate biomarkers based on HPV genotype. (C) Pearson's correlation coefficient analysis plot of p16 score and FEN1 expression in SCC tissues. (D) Pearson's correlation coefficient analysis plot of Ki67 score and FEN1 expression in SCC tissues. For each box plot and Kruskal-Wallis test, the central line represents the median; the outer black lines on the box represent the upper and lower quartiles; dots represent outliers; the black lines are the whiskers, which extend from the interquartile ranges to the maximum values that are not classed as outliers. P-values for each Kruskal-Wallis test are given on the plot. HPV, human papillomavirus; FEN1, flap structure-specific endonuclease 1; SCC, squamous cell carcinoma; ENDOU, poly (U)-specific endoribonuclease; Neg, negative; Pos, positive.

Article Snippet: Antigen retrieval was performed with Tris-EDTA buffer pH 7.8 (Cell Conditioner #1; Ventana Medical System, Inc.) at 95°C for 44 min followed by blocking of endogenous peroxides and proteins with inhibitor CM (Ventana Medical System, Inc.; cat. no. 760-159) at 37°C for 4 min. IHC staining was performed as a fully automated assay in the BenchMark ULTRA automated slide stainer (Ventana Medical System, Inc.) using rabbit polyclonal antibody against FEN1 (1 ng/ml; Atlas antibodies cat. no. HPA006748; http://www.atlasantibodies.com ), ENDOU (2 ng/ml; Atlas antibodies, cat. no. HPA012388; http://www.atlasantibodies.com ), p16 (p16 CINtec ® Histology kit; Roche Diagnostics GmbH; cat no. 06695256001; http://diagnostics.roche.com ) and UltraView DAB IHC Detection kit (cat. no. 760-500; Ventana Medical System, Inc.).

Techniques: Expressing, Infection

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Diagnostic and prognostic performance of FEN1 and ENDOU transcripts. (A) ROC curve analysis for FEN1 in the GSE13380 cervical cancer cohort. (B) ROC curve analysis for ENDOU in the GSE13380 cervical cancer cohort. (C) Overall survival analysis for FEN1 in the TCGA-CESC-SCC cohort. mRNA levels were dichotomized based on the median separation. (D) Overall survival analysis for ENDOU in TCGA-CESC-SCC cohort. mRNA levels were dichotomized based on the median separation. (E) Correlation between FEN1 expression and tumor infiltration signature in TCGA-CESC. (F) Correlation between ENDOU expression and tumor infiltration signature in TCGA-CESC. FEN1, flap structure-specific endonuclease 1; ENDOU, poly (U)-specific endoribonuclease; ROC, receiver operating characteristic; TCGA, The Cancer Genome Atlas; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; SCC, squamous cell carcinoma; AUC, area under the curve; CI, confidence interval; HR, hazard ratio.

Article Snippet: Antigen retrieval was performed with Tris-EDTA buffer pH 7.8 (Cell Conditioner #1; Ventana Medical System, Inc.) at 95°C for 44 min followed by blocking of endogenous peroxides and proteins with inhibitor CM (Ventana Medical System, Inc.; cat. no. 760-159) at 37°C for 4 min. IHC staining was performed as a fully automated assay in the BenchMark ULTRA automated slide stainer (Ventana Medical System, Inc.) using rabbit polyclonal antibody against FEN1 (1 ng/ml; Atlas antibodies cat. no. HPA006748; http://www.atlasantibodies.com ), ENDOU (2 ng/ml; Atlas antibodies, cat. no. HPA012388; http://www.atlasantibodies.com ), p16 (p16 CINtec ® Histology kit; Roche Diagnostics GmbH; cat no. 06695256001; http://diagnostics.roche.com ) and UltraView DAB IHC Detection kit (cat. no. 760-500; Ventana Medical System, Inc.).

Techniques: Diagnostic Assay, Expressing

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Clinical validation of FEN1 and ENDOU in cervical biopsies. (A) Frequency distribution of FEN1 IHC scores in normal and SCC tissues. (B) Frequency distribution of FEN1 IHC scores across SCC grades. (C) Frequency distribution of p16 IHC scores in normal and SCC tissues. (D) Frequency distribution of p16 IHC scores across SCC grades. (E) Frequency distribution of ENDOU immunohistochemistry scores in normal and SCC tissues. (F) IHC analysis of FEN1, ENDOUand p16 expression levels. FEN1, flap structure-specific endonuclease 1;ENDOU, poly (U)-specific endoribonuclease; IHC, immunohistochemistry; SCC, squamous cell carcinoma.

Article Snippet: Antigen retrieval was performed with Tris-EDTA buffer pH 7.8 (Cell Conditioner #1; Ventana Medical System, Inc.) at 95°C for 44 min followed by blocking of endogenous peroxides and proteins with inhibitor CM (Ventana Medical System, Inc.; cat. no. 760-159) at 37°C for 4 min. IHC staining was performed as a fully automated assay in the BenchMark ULTRA automated slide stainer (Ventana Medical System, Inc.) using rabbit polyclonal antibody against FEN1 (1 ng/ml; Atlas antibodies cat. no. HPA006748; http://www.atlasantibodies.com ), ENDOU (2 ng/ml; Atlas antibodies, cat. no. HPA012388; http://www.atlasantibodies.com ), p16 (p16 CINtec ® Histology kit; Roche Diagnostics GmbH; cat no. 06695256001; http://diagnostics.roche.com ) and UltraView DAB IHC Detection kit (cat. no. 760-500; Ventana Medical System, Inc.).

Techniques: Biomarker Discovery, Immunohistochemistry, Expressing

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Association between FEN1 expression and the clinicopathological characteristic.

Article Snippet: Antigen retrieval was performed with Tris-EDTA buffer pH 7.8 (Cell Conditioner #1; Ventana Medical System, Inc.) at 95°C for 44 min followed by blocking of endogenous peroxides and proteins with inhibitor CM (Ventana Medical System, Inc.; cat. no. 760-159) at 37°C for 4 min. IHC staining was performed as a fully automated assay in the BenchMark ULTRA automated slide stainer (Ventana Medical System, Inc.) using rabbit polyclonal antibody against FEN1 (1 ng/ml; Atlas antibodies cat. no. HPA006748; http://www.atlasantibodies.com ), ENDOU (2 ng/ml; Atlas antibodies, cat. no. HPA012388; http://www.atlasantibodies.com ), p16 (p16 CINtec ® Histology kit; Roche Diagnostics GmbH; cat no. 06695256001; http://diagnostics.roche.com ) and UltraView DAB IHC Detection kit (cat. no. 760-500; Ventana Medical System, Inc.).

Techniques: Expressing

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Diagnostic accuracy of FEN1 staining and comparator tests.

Article Snippet: Antigen retrieval was performed with Tris-EDTA buffer pH 7.8 (Cell Conditioner #1; Ventana Medical System, Inc.) at 95°C for 44 min followed by blocking of endogenous peroxides and proteins with inhibitor CM (Ventana Medical System, Inc.; cat. no. 760-159) at 37°C for 4 min. IHC staining was performed as a fully automated assay in the BenchMark ULTRA automated slide stainer (Ventana Medical System, Inc.) using rabbit polyclonal antibody against FEN1 (1 ng/ml; Atlas antibodies cat. no. HPA006748; http://www.atlasantibodies.com ), ENDOU (2 ng/ml; Atlas antibodies, cat. no. HPA012388; http://www.atlasantibodies.com ), p16 (p16 CINtec ® Histology kit; Roche Diagnostics GmbH; cat no. 06695256001; http://diagnostics.roche.com ) and UltraView DAB IHC Detection kit (cat. no. 760-500; Ventana Medical System, Inc.).

Techniques: Diagnostic Assay, Staining

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Potential function of FEN1 and ENDOU in cervical carcinogenesis. (A) Pearson's correlation coefficient analysis plot of G 2 /M score and FEN1 expression in SCC tissues. (B) FEN1 concept network for prediction of gene function. (C) Ridge plot illustrating GO enrichment in cervical tumors expressing high levels of FEN1. (D) Correlation matrix between FEN1 and phase-specific cyclins, CDKs and regulators. (E) Correlation matrix between ENDOU and different cytokeratins. (F) Ridge plot illustrating Gene Set Enrichment Analysis, GO enrichment in cervical tumors expressing high levels of ENDOU. FEN1, flap structure-specific endonuclease 1; ENDOU, poly (U)-specific endoribonuclease; SCC, squamous cell carcinoma; GO, Gene Ontology.

Article Snippet: Antigen retrieval was performed with Tris-EDTA buffer pH 7.8 (Cell Conditioner #1; Ventana Medical System, Inc.) at 95°C for 44 min followed by blocking of endogenous peroxides and proteins with inhibitor CM (Ventana Medical System, Inc.; cat. no. 760-159) at 37°C for 4 min. IHC staining was performed as a fully automated assay in the BenchMark ULTRA automated slide stainer (Ventana Medical System, Inc.) using rabbit polyclonal antibody against FEN1 (1 ng/ml; Atlas antibodies cat. no. HPA006748; http://www.atlasantibodies.com ), ENDOU (2 ng/ml; Atlas antibodies, cat. no. HPA012388; http://www.atlasantibodies.com ), p16 (p16 CINtec ® Histology kit; Roche Diagnostics GmbH; cat no. 06695256001; http://diagnostics.roche.com ) and UltraView DAB IHC Detection kit (cat. no. 760-500; Ventana Medical System, Inc.).

Techniques: Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Adenomatous Polyposis Coli Interacts with Flap Endonuclease 1 to Block Its Nuclear Entry and Function 1

doi:

Figure Lengend Snippet: Oligonucleotide Sequences for Different Primers and Substrates for Fen1.

Article Snippet: Binding was detected using mouse anti-Fen1 antibody (Novus Biologicals, Littleton, CO).

Techniques: Mutagenesis, Activity Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Adenomatous Polyposis Coli Interacts with Flap Endonuclease 1 to Block Its Nuclear Entry and Function 1

doi:

Figure Lengend Snippet: Colocalization of APC(1-1309) and Fen1 in MB231 cells after treatment with B[a]P. (A and B) Cells were transfected with flag-pAPC(1–1309) or flag-pAPC(1–1309mut) plasmids for 24 hours. MDA-MB231 cells were stained for APC (Alexa-594, red) and Fen1 (Alexa-488, green). The localization of flag-APC and Fen1 was determined by fluorescence microscopy. Images were merged to determine the colocalization of these proteins. Data are representative of three independent experiments. (C) Immunofluorescence analysis of wildtype APC and Fen1 colocalization in MB231 cells treated with B[a]P. Cells were treated with 25 µMB[a]P for 24 hours. The colocalization of endogenous APC and Fen1 was determined by fluorescence microscopy. Images were merged to determine the colocalization of these proteins. Data are representative of three independent experiments.

Article Snippet: Binding was detected using mouse anti-Fen1 antibody (Novus Biologicals, Littleton, CO).

Techniques: Transfection, Staining, Fluorescence, Microscopy, Immunofluorescence

Journal: Neoplasia (New York, N.Y.)

Article Title: Adenomatous Polyposis Coli Interacts with Flap Endonuclease 1 to Block Its Nuclear Entry and Function 1

doi:

Figure Lengend Snippet: Colocalization of wild-type APC and Fen1 in HCT-116 cells after treatment with MMS. (A) HCT-116 and HCT-116-APC(KD) cells were treated with 500 µM MMS for 24 hours. Cells were stained for APC (Alexa-594, red), Fen1 (Alexa-488, green), and heterochromatin (DAPI, blue). The localization of flag-APC, Fen1, and heterochromatin was determined by fluorescence microscopy. Images were merged to determine the colocalization of these proteins. Data are representative of three independent experiments. (B) Western blot analysis. Cells were treated with 0, 50, 100, 250, and 500 µM MMS for 24 hours. The cytoplasmic and nuclear fractions were prepared and used for the determination of APC and Fen1 levels. β-Actin and lamin B levels were determined for protein loading normalization and for identifying potential cross-contamination of the cytoplasmic and nuclear fraction preparations. Data are representative of two independent experiments.

Article Snippet: Binding was detected using mouse anti-Fen1 antibody (Novus Biologicals, Littleton, CO).

Techniques: Staining, Fluorescence, Microscopy, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Adenomatous Polyposis Coli Interacts with Flap Endonuclease 1 to Block Its Nuclear Entry and Function 1

doi:

Figure Lengend Snippet: APC knockdown increases Fen1 levels and activity in the nucleus. APC knockdown in MB468 breast cancer cells was accomplished by transfection with pSiRNA-APC, as shown in Figure 1. (A) The cytoplasmic and nuclear fractions were prepared and used for the determination of APC and Fen1 levels. GAPDH and lamin B protein levels were determined for protein loading normalization and for identifying potential cross-contamination of the cytoplasmic and nuclear fraction preparations. (B and C) Quantitative analysis of APC and Fen1 proteins levels. (D) The nuclear extract was also used for the determination of LP-BER. Lanes 1 and 2 show 32P-labeled 63-mer F-DNA and APE1 incised 23-mer product, respectively. Data shown are representative of two independent experiments.

Article Snippet: Binding was detected using mouse anti-Fen1 antibody (Novus Biologicals, Littleton, CO).

Techniques: Knockdown, Activity Assay, Transfection, Labeling

Journal: Neoplasia (New York, N.Y.)

Article Title: Adenomatous Polyposis Coli Interacts with Flap Endonuclease 1 to Block Its Nuclear Entry and Function 1

doi:

Figure Lengend Snippet: Interaction of APC and Fen1 protein fragments. APCwt and APC(I-A,Y-A) protein fragments were immobilized on a PVDF membrane and incubated with either wild-type or fragments of Fen1 protein as indicated. Equal amounts of TNT-synthesized proteins were used. Data are representative of three different determinations.

Article Snippet: Binding was detected using mouse anti-Fen1 antibody (Novus Biologicals, Littleton, CO).

Techniques: Membrane, Incubation, Synthesized

Journal: Neoplasia (New York, N.Y.)

Article Title: Adenomatous Polyposis Coli Interacts with Flap Endonuclease 1 to Block Its Nuclear Entry and Function 1

doi:

Figure Lengend Snippet: Interaction of APC with Fen1 by a yeast two-hybrid system. (A) Structure of APC and Fen1 constructs. The mutated amino acid residues I and Y with A are shown with italics. (B) Yeast two-hybrid analysis of APC and Fen1 interaction. (C) Far-Western analysis of APC and Fen1 showing that the D181N mutation in Fen1 reduces APC binding. Data are representative of three independent experiments.

Article Snippet: Binding was detected using mouse anti-Fen1 antibody (Novus Biologicals, Littleton, CO).

Techniques: Construct, Western Blot, Mutagenesis, Binding Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Adenomatous Polyposis Coli Interacts with Flap Endonuclease 1 to Block Its Nuclear Entry and Function 1

doi:

Figure Lengend Snippet: Interaction of APC with NLS domains of Fen1. (A) Far-Western analysis of the interaction of APC with Fen1wt, Fen1 (NLS1mut), and Fen1 (NLS2mut) protein fragments. APCwt and APC(I-A,Y-A) peptides were immobilized on a PVDF membrane and incubated with either wild-type or fragments of Fen1 protein as indicated. (B) Percent binding of APCwt with Fen1 wild-type and mutant proteins. Percent binding was determined by using the density of APC and Fen1 wild-type binding band as 100% binding and comparing Fen1 mutant proteins and APC binding to the density of the APC and Fen1 wild-type band [(density of APCwt and Fen1 mutant binding/density of APCwt and Fen1 wild-type) x 100 = percent bound)]. The experiment was repeated twice.

Article Snippet: Binding was detected using mouse anti-Fen1 antibody (Novus Biologicals, Littleton, CO).

Techniques: Western Blot, Membrane, Incubation, Binding Assay, Mutagenesis

Journal: Neoplasia (New York, N.Y.)

Article Title: Adenomatous Polyposis Coli Interacts with Flap Endonuclease 1 to Block Its Nuclear Entry and Function 1

doi:

Figure Lengend Snippet: Fen1(NLS2mut) retains 5′-flap endonuclease and 5′-3′ exonuclease activities. (A) Schematic of protocol for the pull-down of Fen1 proteins from the TNT reaction mixture. (B) Western blot analysis of Fen1 proteins synthesized by TNT. (C) Autoradiogram depicting 5′-flap endonuclease activity. Equal amounts of TNT-synthesized proteins were used in these assays. Purified Fen1wt protein at 0.2 nM was used as a positive control. A 11-nt cleaved endonuclease product is shown with an arrow. (D) Autoradiogram depicting 5′-3′ exonuclease activity. A 1-nt cleaved exonuclease product is shown with an arrow. Equal amounts of TNT-synthesized proteins were used in these assays. Data are representative of three different determinations.

Article Snippet: Binding was detected using mouse anti-Fen1 antibody (Novus Biologicals, Littleton, CO).

Techniques: Western Blot, Synthesized, Activity Assay, Purification, Positive Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Adenomatous Polyposis Coli Interacts with Flap Endonuclease 1 to Block Its Nuclear Entry and Function 1

doi:

Figure Lengend Snippet: APC/Fen1 interaction in human breast tumor tissues. Normal and breast tumor tissues were stained for APC (Alexa-594, red), Fen1 (Alexa-488, green), and nuclei (DAPI, blue). The localization of APC and Fen1 was determined by fluorescence microscopy. Images were merged to observe colocalization.

Article Snippet: Binding was detected using mouse anti-Fen1 antibody (Novus Biologicals, Littleton, CO).

Techniques: Staining, Fluorescence, Microscopy

Journal: BMC Cancer

Article Title: YY1 suppresses FEN1 over-expression and drug resistance in breast cancer

doi: 10.1186/s12885-015-1043-1

Figure Lengend Snippet: Identification of YY1 as a potential transcription regulator for FEN1. A . Top 10 hits of the transcription factors (TFs) that were predicted by TF Research Web sites: Match1.0-public, PROMO, and TFSEARCH. B . The oligo probes were designed to cover different regions of the predicted FEN1 promoter. Probes a, b, and c correspond to the region −290 to −230, −150 to −90, and −60 to 0, respectively. C . The silver staining image of oligo-pulled-down assays using HeLa cell extracts. b SNP : probe b with three SNP sites, r: a probe with random DNA sequences. The unique band, which is indicated by a box, was subjected to MS analysis. D . Top 10 hits of the MS analysis of the unique protein band as specified in Panel C .

Article Snippet: The antibodies used in our studies were the rabbit polyclonal anti-YY1 antibody (Santa Cruz), the rabbit monoclonal anti-FEN1 antibody (Novus Biologicals, Littleton, CO, USA), the Horseradish peroxidase (HRP)-conjugated anti-GAPDH (GenScript, China), and the Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Pierce, Rockford, IL, USA).

Techniques: Silver Staining

Journal: BMC Cancer

Article Title: YY1 suppresses FEN1 over-expression and drug resistance in breast cancer

doi: 10.1186/s12885-015-1043-1

Figure Lengend Snippet: YY1 binds to the conserved YY1 binding motif in the FEN1 promoter region. A . Sequence alignment of the conserved YY1 binding motif in different proteins. B . EMSA analysis of YY1 binding to the YY1 binding motif in the FEN1 promoter. Recombinant YY1 was incubated with different biotin-labeled DNA probes. The sequences of the Probe N, Probe P, WT FEN1and MUT FEN1 can be found in Additional file : Table S6. The free probe and YY1/DNA complex were resolved in 5% native PAGE. C . EMSA assay on YY1 and FEN1 oligo in the presence of non-specific IgG or the anti-YY1 antibody. D . ChIP analysis of YY1 binding to the FEN1 promoter region. Specific YY1-bound DNA in MCF7 cell extracts was pulled down by an anti-YY1 antibody. The YY1-bound FEN1 sequence was amplified by PCR. The sequence for the FEN1 promoter specific primer can be found in the Additional file : Table S6 as FEN1 (YY1). The PCR product was analyzed by 1% agarose electrophoresis.

Article Snippet: The antibodies used in our studies were the rabbit polyclonal anti-YY1 antibody (Santa Cruz), the rabbit monoclonal anti-FEN1 antibody (Novus Biologicals, Littleton, CO, USA), the Horseradish peroxidase (HRP)-conjugated anti-GAPDH (GenScript, China), and the Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Pierce, Rockford, IL, USA).

Techniques: Binding Assay, Sequencing, Recombinant, Incubation, Labeling, Clear Native PAGE, Amplification, Electrophoresis

Journal: BMC Cancer

Article Title: YY1 suppresses FEN1 over-expression and drug resistance in breast cancer

doi: 10.1186/s12885-015-1043-1

Figure Lengend Snippet: Overexpression of YY1 inhibits FEN1 promoter-driven protein expression. A . YY1 was overexpressed in 293 T cells and its impact on the FEN1 protein level was evaluated by western blot using the anti-Flag or anti-FEN1 antibody. B . The pCMV-Flag-YY1 expression vector and the pGL4.0-FEN1 promoter-EGFP vector, or pGL4.0 EGFP vector was co-transfected into 293 T cells. The EGFP expression was detected by semi-quantitative PCR (Upper panel) and quantitative PCR (lower panel). C . The overexpression of Flag-tagged YY1 was confirmed by western blot using the anti-Flag antibody. D and E . EGFP protein levels with or without YY1 overexpression was measured by FACS. Panel D shows the representative FACS images. Panel E is the quantification of FACS. Values are means ± s.d. of four independent experiments. p value was calculated by the two-tail student’s t-test. F . Knockdown of YY1 in 293 T (Left Panel) and MCF7 (right panel) cells. The YY1 and FEN1 expression was measured by quantitative PCR. The mRNA level was normalized with corresponding mRNA level of GADPH, and the normalized mRNA level of YY1 or FEN1 in the cells treated with control siRNA was arbitrarily set as 1. Values are means ± s.d. of three independent experiments. p value was calculated by the two-tail student’s t-test.

Article Snippet: The antibodies used in our studies were the rabbit polyclonal anti-YY1 antibody (Santa Cruz), the rabbit monoclonal anti-FEN1 antibody (Novus Biologicals, Littleton, CO, USA), the Horseradish peroxidase (HRP)-conjugated anti-GAPDH (GenScript, China), and the Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Pierce, Rockford, IL, USA).

Techniques: Over Expression, Expressing, Western Blot, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Knockdown, Control

Journal: BMC Cancer

Article Title: YY1 suppresses FEN1 over-expression and drug resistance in breast cancer

doi: 10.1186/s12885-015-1043-1

Figure Lengend Snippet: DNA damaging agents MMC and Taxol inhibit YY1 expression but induce FEN1 expression. A . and B . YY1 and FEN1 expression in MDA-MB-231 breast cancer cell line in response to the MMC and Taxol treatment. The mRNA level (A) and protein level (B) were measured by quantitative PCR and Western blot. The left panel in B showed the quantification of Western blot results. All experiments were independent carried out at least three times. C . Analysis of YY1 binding to the FEN1 promoter in response to the MMC treatment. Cells were treated with MMC, and the level of YY1-bound FEN1 promoter was evaluated by the ChIP assay. The lowest DNA staining density is arbitrarily set as 1. D . western blot confirmed the overexpression of Flag-tagged YY1 in 293 T cells. The β-actin(ACTB) was used as control. E . and F . The survivorship of 293 T cell and 293 T cell harboring a YY1 expression plasmid, pCMV-Flag-YY1, or the empty vector, under treatment of mytomycine C (MMC) (Panel E) or taxol (Panel F) . In both panels, the cells were treated with indicated concentrations of MMC or taxol for 48 hours. The number of survival cells was counted. The survival rate of the untreated cells with or without YY1 overexpression was arbitrarily set as 1.

Article Snippet: The antibodies used in our studies were the rabbit polyclonal anti-YY1 antibody (Santa Cruz), the rabbit monoclonal anti-FEN1 antibody (Novus Biologicals, Littleton, CO, USA), the Horseradish peroxidase (HRP)-conjugated anti-GAPDH (GenScript, China), and the Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Pierce, Rockford, IL, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay, Staining, Over Expression, Control, Plasmid Preparation

Journal: BMC Cancer

Article Title: YY1 suppresses FEN1 over-expression and drug resistance in breast cancer

doi: 10.1186/s12885-015-1043-1

Figure Lengend Snippet: FEN1 gene expression in response to chemotherapeutic drug and DNA damage agent treatments. A . Macro-image of hybridization of the 32 -P-labeled FEN1 ORF DNA fragment with the expression array, which contains cDNA from different cells lines treated with different DNA-damaging agents. In the control hybridization, 32 -P-labeled ubiquitin ORF DNA fragment was used . B . The relative fold changes of the density of the hybridized spots. All FEN1 hybridization signals were normalized with corresponding ubiquitin signals. In each cell line, the normalized signal in the untreated control sample was arbitrarily set as 1, and the fold change was calculated by comparing the normalized signal in a specific sample to that of the untreated control sample.

Article Snippet: The antibodies used in our studies were the rabbit polyclonal anti-YY1 antibody (Santa Cruz), the rabbit monoclonal anti-FEN1 antibody (Novus Biologicals, Littleton, CO, USA), the Horseradish peroxidase (HRP)-conjugated anti-GAPDH (GenScript, China), and the Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Pierce, Rockford, IL, USA).

Techniques: Gene Expression, Hybridization, Labeling, Expressing, Control, Ubiquitin Proteomics

Journal: BMC Cancer

Article Title: YY1 suppresses FEN1 over-expression and drug resistance in breast cancer

doi: 10.1186/s12885-015-1043-1

Figure Lengend Snippet: Associations between FEN1, YY1 protein, or their combination and OS or DFS. A . FEN1 Kaplan Meier survival plot with breast cancer patient cohort in the Ivshina data base. The black line indicates FEN1 high expression while the red line indicates FEN1 low expression (‘high’ and ‘low’ determined by median expression). Patients with FEN1 high expression: 132; patients with FEN1 low expression: 117, Log-rank p = 0.0007. B . Low expression of YY1 and high expression of FEN1 and OS in the CMU cohort. The black line indicates YY1 low but FEN1 high expression and red line indicates other types. Patients with YY1 low but FEN1 high expression: 154; patients with other types: 113, Log-rank p = 0.027. C . Low expression of YY1 and high expression of FEN1 and DFS in the CMU cohort. The black line indicates YY1 low but FEN1 high expression and red line indicates other types. Patients with YY1 low but FEN1 high expression: 154; patients with other type: 113, Log-rank p = 0.048.

Article Snippet: The antibodies used in our studies were the rabbit polyclonal anti-YY1 antibody (Santa Cruz), the rabbit monoclonal anti-FEN1 antibody (Novus Biologicals, Littleton, CO, USA), the Horseradish peroxidase (HRP)-conjugated anti-GAPDH (GenScript, China), and the Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Pierce, Rockford, IL, USA).

Techniques: Expressing