Journal: International Journal of Oncology

Article Title: High levels of human epididymis protein 4 mRNA and protein expression are associated with chemoresistance and a poor prognosis in pancreatic cancer

doi: 10.3892/ijo.2020.5147

Figure Lengend Snippet: Identification of chemotherapy resistance molecules. (A and B) Results of NGS analysis of (A) GEM and (B) GEM + nab-PTX administration. The genes are shown on the horizontal axis, and the treatment resistance score is shown on the vertical axis. Treatment resistance score was calculated by the ratio of NE value (treated group/control group) multiplied by the difference in NE value (treated group-control group). In both cases (A and B), HE4 was upregulated in the treatment group. (C and D) The NE value of HE4 mRNA. The NE value ratio of HE4 expression between the treatment and control groups was often >1.0 in PDXs treated with GEM or GEM plus nab-PTX. HE4, human epididymis protein 4; PDX, patient-derived xenograft; GEM, gemcitabine; nab-PTX, nab-paclitaxel.

Article Snippet: For transient overexpression experiments, an expression plasmid vector for HE4 (pCMV6-AC-HE4-GFP tag plasmid, NM_006418) was obtained from OriGene Technologies, Inc. and an empty vector (pCMV6-AC-GFP) was adopted for control transfection.

Techniques: Expressing, Derivative Assay

Journal: International Journal of Oncology

Article Title: High levels of human epididymis protein 4 mRNA and protein expression are associated with chemoresistance and a poor prognosis in pancreatic cancer

doi: 10.3892/ijo.2020.5147

Figure Lengend Snippet: Kaplan-Meier plots demonstrating the correlation between HE4 mRNA expression and patient survival in TCGA database for pancreatic cancer (n=166). Red line, high expression (n=34); blue line, low expression (n=132). HE4, human epididymis protein 4. The asterisk (*) indicates a statistically significant difference (P<0.05).

Article Snippet: For transient overexpression experiments, an expression plasmid vector for HE4 (pCMV6-AC-HE4-GFP tag plasmid, NM_006418) was obtained from OriGene Technologies, Inc. and an empty vector (pCMV6-AC-GFP) was adopted for control transfection.

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: High levels of human epididymis protein 4 mRNA and protein expression are associated with chemoresistance and a poor prognosis in pancreatic cancer

doi: 10.3892/ijo.2020.5147

Figure Lengend Snippet: Expression of HE4 and cell viability assays of cell lines. (A) The levels of HE4 gene expression in 9 tumor cell lines (SK-N-AS, KATOIII, ASPC-1, PANC-1, SUIT-2, MIA-Paca2, IMR-32, HCT116 and HeLa) and 2 non-cancerous cell lines (HUVECs and HRGECs) measured by RT-qPCR. Endogenous HE4 expression levels differed among cell lines. * P<0.05. (B) Cell viability assays of tumor cell lines in response to GEM treatment. When the concentration of GEM was increased, the relative viable cell numbers tended to be higher in the cell lines in which expression of endogenous HE4 was relatively higher. Cell lines with different lowercase letters (a-g) indicate statistically significant differences at each GEM concentration. a P<0.05 vs. b-g; b P<0.05 vs. c-g; c P<0.05 vs. d-g; d P<0.05 vs. e-g; e P<0.05 vs. f and g; and f P<0.05 vs. g. HE4, human epididymis protein 4; GEM, gemcitabine; HUVECs, human umbilical vein endothelial cells; HRGECs, human renal glomerular endothelial cells.

Article Snippet: For transient overexpression experiments, an expression plasmid vector for HE4 (pCMV6-AC-HE4-GFP tag plasmid, NM_006418) was obtained from OriGene Technologies, Inc. and an empty vector (pCMV6-AC-GFP) was adopted for control transfection.

Techniques: Expressing, Quantitative RT-PCR, Concentration Assay

Journal: International Journal of Oncology

Article Title: High levels of human epididymis protein 4 mRNA and protein expression are associated with chemoresistance and a poor prognosis in pancreatic cancer

doi: 10.3892/ijo.2020.5147

Figure Lengend Snippet: Viability assay of 293 cells. (A) Expression of HE4 and control vectors in 293 cells followed by treatment with GEM after 24 h (day 1). MTT assay was performed 48 h following treatment with GEM at various concentrations (day 3). (B and C) Histograms representing the rates of change of each measured OD value of control vector and HE4-expressing 293 cells. * P<0.0007, control vector vs. HE4-expressing cells at each GEM concentration, as determined by two-way ANOVA for multiple comparisons with the Bonferroni post hoc test. HE4, human epididymis protein 4; GEM, gemcitabine.

Article Snippet: For transient overexpression experiments, an expression plasmid vector for HE4 (pCMV6-AC-HE4-GFP tag plasmid, NM_006418) was obtained from OriGene Technologies, Inc. and an empty vector (pCMV6-AC-GFP) was adopted for control transfection.

Techniques: Viability Assay, Expressing, MTT Assay, Plasmid Preparation, Concentration Assay

Journal: International Journal of Oncology

Article Title: High levels of human epididymis protein 4 mRNA and protein expression are associated with chemoresistance and a poor prognosis in pancreatic cancer

doi: 10.3892/ijo.2020.5147

Figure Lengend Snippet: Association between HE4 expression and patient prognosis. (A) IHC analysis of HE4 in patient tissues (magnification, ×100). Images are of the same sample tissue sections and representing the intensity of stain. Images in the left panel show H&E staining, and those in the right indicate IHC detection of HE4. (B) The criteria for evaluation of HE4 expression levels. The intensity of stain and percentage criteria are designated. (C) Kaplan-Meier survival analysis in pancreatic cancer patients (n=80). Overall survival is shown in accordance with the protein expression level of HE4. The red line represents the group of patients with high HE4 expression (n=44), and the blue line indicates those with low HE4 expression (n=36). HE4, human epididymis protein 4.

Article Snippet: For transient overexpression experiments, an expression plasmid vector for HE4 (pCMV6-AC-HE4-GFP tag plasmid, NM_006418) was obtained from OriGene Technologies, Inc. and an empty vector (pCMV6-AC-GFP) was adopted for control transfection.

Techniques: Expressing, Staining

Journal: International Journal of Oncology

Article Title: High levels of human epididymis protein 4 mRNA and protein expression are associated with chemoresistance and a poor prognosis in pancreatic cancer

doi: 10.3892/ijo.2020.5147

Figure Lengend Snippet: Association between HE4 expression and clinicopathological features in 80 cases of pancreatic cancer.

Article Snippet: For transient overexpression experiments, an expression plasmid vector for HE4 (pCMV6-AC-HE4-GFP tag plasmid, NM_006418) was obtained from OriGene Technologies, Inc. and an empty vector (pCMV6-AC-GFP) was adopted for control transfection.

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: High levels of human epididymis protein 4 mRNA and protein expression are associated with chemoresistance and a poor prognosis in pancreatic cancer

doi: 10.3892/ijo.2020.5147

Figure Lengend Snippet: Univariate and multivariate analyses of prognostic factor for overall survival in 80 pancreatic cancer patients.

Article Snippet: For transient overexpression experiments, an expression plasmid vector for HE4 (pCMV6-AC-HE4-GFP tag plasmid, NM_006418) was obtained from OriGene Technologies, Inc. and an empty vector (pCMV6-AC-GFP) was adopted for control transfection.

Techniques:

Journal: PLOS ONE

Article Title: The NF-κB-HE4 axis: A novel regulator of HE4 secretion in ovarian cancer

doi: 10.1371/journal.pone.0314564

Figure Lengend Snippet: (A) TNFα enhanced the nuclear localization of p65 and p50 in A2780 cells. A2780 and OVCAR-3 cells were stimulated with TNFα (30 ng/mL) for 5 hours. Cells were lysed, and cytoplasmic and nuclear fractions were isolated, followed by immunoblot analysis using antibodies against GAPDH (a cytoplasmic marker) and lamin A/C (a nuclear marker), as well as against p65, and p50. The expression levels of p65 and p50 in nuclear fraction upon TNFα stimulation were determined relative to Lamin A/C expression (top). (B) p65 knockdown abolished TNFα induced HE4 secretion in A2780 cells. A2780 cells were transfected with siRNAs against p65 or with non-targeting control. Following transfection cells were exposed to TNFα treatment (30 ng/mL) for 48 hours. Afterward, cell culture medium was collected. HE4 levels were determined using HE4 ELISA. (C) p65 expression promotes HE4 expression in OVCAR-3 cells. Cell culture medium from OVCAR-3 cells transfected for 72 hours with empty or p65 expression vector was analyzed for HE4 levels using HE4 ELISA (left). Simultaneously, the cell population for each condition was assessed via the MTS assay (right).

Article Snippet: Santa Cruz Biotechnology siRNAs for control (cat. No. sc-37007) and HE4 (cat. No. sc-43826); Vectors: empty (addgene, cat. No. 24165), p65 (addgene, cat. No. 21966), IKK2-EE (addgene, cat. No.11105); antibodies: p53 (Santa Cruz Biotechnology cat. No. sc-126), HE4 (OriGene cat. No. UM870019), p50 (BioLegend cat. No. 603901), P-c-Jun (BioLegend cat. No. 605551).

Techniques: Isolation, Western Blot, Marker, Expressing, Knockdown, Transfection, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, MTS Assay

Journal: PLOS ONE

Article Title: The NF-κB-HE4 axis: A novel regulator of HE4 secretion in ovarian cancer

doi: 10.1371/journal.pone.0314564

Figure Lengend Snippet: (A) Expression of constitutively active IKK2 mutant (IKK2-EE) led to the phosphorylation of Iκβα. 2008 cells were transfected with either IKK2-EE or an empty vector for 24 hours. Cells were lysed, and protein expression was assessed via immunoblot analysis. Phosphorylated Iκβα levels were quantified relative to total Iκβα. (B) IKK2 inhibitor TPCA-1 reduced HE4 expression. 2008 cells were treated with TPCA-1 at a range of concentrations for 24 hours. HE4 levels in culture medium were determined using HE4 ELISA (left). Simultaneously, the cell population for each condition was assessed via the MTS assay (right) (C-E) Expression of p65 and IKK2-EE promoted HE4 expression in ovarian cancer cell lines. A2780, OVCAR-3, and 2008 cells were transfected with p65, IKK2-EE, or empty vector for 72 hours. Afterward, HE4 levels in culture medium were determined using HE4 ELISA.

Article Snippet: Santa Cruz Biotechnology siRNAs for control (cat. No. sc-37007) and HE4 (cat. No. sc-43826); Vectors: empty (addgene, cat. No. 24165), p65 (addgene, cat. No. 21966), IKK2-EE (addgene, cat. No.11105); antibodies: p53 (Santa Cruz Biotechnology cat. No. sc-126), HE4 (OriGene cat. No. UM870019), p50 (BioLegend cat. No. 603901), P-c-Jun (BioLegend cat. No. 605551).

Techniques: Expressing, Mutagenesis, Phospho-proteomics, Transfection, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, MTS Assay

Journal: PLOS ONE

Article Title: The NF-κB-HE4 axis: A novel regulator of HE4 secretion in ovarian cancer

doi: 10.1371/journal.pone.0314564

Figure Lengend Snippet: A2780 cells were treated with TNFα (30 ng/mL) for 24 hours. OVCAR-3 and 2008 cells were transfected with p65, IKK2-EE, or empty vector for 24 hours. Cells were lysed, and isolated RNAs were analyzed for HE4 (A) or TNF (B) via quantitative reverse transcription PCR, as described in “Materials and Methods”.

Article Snippet: Santa Cruz Biotechnology siRNAs for control (cat. No. sc-37007) and HE4 (cat. No. sc-43826); Vectors: empty (addgene, cat. No. 24165), p65 (addgene, cat. No. 21966), IKK2-EE (addgene, cat. No.11105); antibodies: p53 (Santa Cruz Biotechnology cat. No. sc-126), HE4 (OriGene cat. No. UM870019), p50 (BioLegend cat. No. 603901), P-c-Jun (BioLegend cat. No. 605551).

Techniques: Transfection, Plasmid Preparation, Isolation, Reverse Transcription

Journal: PLOS ONE

Article Title: The NF-κB-HE4 axis: A novel regulator of HE4 secretion in ovarian cancer

doi: 10.1371/journal.pone.0314564

Figure Lengend Snippet: TNFα promoted HE4 secretion through the NF-kB signaling pathway (A) A2780 cells were incubated with TPCA-1 (25 μM, an IKK2 inhibitor), BIRB-769 (1 μM, a p38 inhibitor), JNK-IN-8 (1 μM, a JNK inhibitor), or CI-1040 (20 μM, a MEK inhibitor) for 1 hour, after which cells were subjected to TNFα treatment (30 ng/mL) for 15 minutes. Subsequently, protein expression and phosphorylation were assessed via immunoblot analysis using target-specific antibodies, with α-tubulin serving as a loading control. (B) A2780 cells underwent the same inhibitor treatment as in (A), after which cells were treated with TNFα (30 ng/mL) for 24 hours. HE4 levels in culture medium were determined using HE4 ELISA. (C) A2780 cells were transfected with a construct encoding luciferase under the control of an NF-κB response element or HE4 promoter construct (HE4-652), and luciferase activity was assessed following TNFα (30 ng/mL) or IL-1β (10 ng/mL) treatment for 7 hours. (D) Same as (C), but HCH-1 cells were employed.

Article Snippet: Santa Cruz Biotechnology siRNAs for control (cat. No. sc-37007) and HE4 (cat. No. sc-43826); Vectors: empty (addgene, cat. No. 24165), p65 (addgene, cat. No. 21966), IKK2-EE (addgene, cat. No.11105); antibodies: p53 (Santa Cruz Biotechnology cat. No. sc-126), HE4 (OriGene cat. No. UM870019), p50 (BioLegend cat. No. 603901), P-c-Jun (BioLegend cat. No. 605551).

Techniques: Incubation, Expressing, Phospho-proteomics, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Transfection, Construct, Luciferase, Activity Assay

Journal: PLOS ONE

Article Title: The NF-κB-HE4 axis: A novel regulator of HE4 secretion in ovarian cancer

doi: 10.1371/journal.pone.0314564

Figure Lengend Snippet: (A) Expression of constitutively active IKK2 mutant (IKK2-EE) led to the phosphorylation of Iκβα. 2008 cells were transfected with either IKK2-EE or an empty vector for 24 hours. Cells were lysed, and protein expression was assessed via immunoblot analysis. Phosphorylated Iκβα levels were quantified relative to total Iκβα. (B) IKK2 inhibitor TPCA-1 reduced HE4 expression. 2008 cells were treated with TPCA-1 at a range of concentrations for 24 hours. HE4 levels in culture medium were determined using HE4 ELISA (left). Simultaneously, the cell population for each condition was assessed via the MTS assay (right) (C-E) Expression of p65 and IKK2-EE promoted HE4 expression in ovarian cancer cell lines. A2780, OVCAR-3, and 2008 cells were transfected with p65, IKK2-EE, or empty vector for 72 hours. Afterward, HE4 levels in culture medium were determined using HE4 ELISA.

Article Snippet: Santa Cruz Biotechnology siRNAs for control (cat. No. sc-37007) and HE4 (cat. No. sc-43826); Vectors: empty (addgene, cat. No. 24165), p65 (addgene, cat. No. 21966), IKK2-EE (addgene, cat. No.11105); antibodies: p53 (Santa Cruz Biotechnology cat. No. sc-126), HE4 (OriGene cat. No. UM870019), p50 (BioLegend cat. No. 603901), P-c-Jun (BioLegend cat. No. 605551).

Techniques: Expressing, Mutagenesis, Phospho-proteomics, Transfection, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, MTS Assay

Journal: PLOS ONE

Article Title: The NF-κB-HE4 axis: A novel regulator of HE4 secretion in ovarian cancer

doi: 10.1371/journal.pone.0314564

Figure Lengend Snippet: A2780 cells were treated with TNFα (30 ng/mL) for 24 hours. OVCAR-3 and 2008 cells were transfected with p65, IKK2-EE, or empty vector for 24 hours. Cells were lysed, and isolated RNAs were analyzed for HE4 (A) or TNF (B) via quantitative reverse transcription PCR, as described in “Materials and Methods”.

Article Snippet: Santa Cruz Biotechnology siRNAs for control (cat. No. sc-37007) and HE4 (cat. No. sc-43826); Vectors: empty (addgene, cat. No. 24165), p65 (addgene, cat. No. 21966), IKK2-EE (addgene, cat. No.11105); antibodies: p53 (Santa Cruz Biotechnology cat. No. sc-126), HE4 (OriGene cat. No. UM870019), p50 (BioLegend cat. No. 603901), P-c-Jun (BioLegend cat. No. 605551).

Techniques: Transfection, Plasmid Preparation, Isolation, Reverse Transcription

Journal: PLOS ONE

Article Title: The NF-κB-HE4 axis: A novel regulator of HE4 secretion in ovarian cancer

doi: 10.1371/journal.pone.0314564

Figure Lengend Snippet: (A) 2008 cells were co-transfected with IKK2-EE expression vector and HE4 siRNAs (sourced from two different vendors) for 48 hours. Cells were lysed, and protein expression of putative NF-kB targets was measured via immunoblot analysis. Heat map represents the relative expression of each NF-kB target evaluated by densitometric analysis. GAPDH served as a loading control for normalization. (B) 2008 cells were transfected with HE4 siRNAs from three different vendors (48 or 72 hours). Expression of HE4, α 5 -integrin, and α-tubulin was measured via immunoblot analysis. (C)The effects of HE4 knockdown on adhesion molecules. 2008 cells were transfected with HE4 siRNA. Protein extracts were prepared and subjected to immunoblot analysis against the proteins as indicated. (D) as in (C) but other ovarian (HCH-1 and Caov-3) and non-ovarian (BxPC-3, pancreatic) cancer cell lines were employed. (E) HE4 knockdown inhibited cell adhesion to fibronectin. Cell adhesion of 2008 cells transfected with HE4 siRNA (72 hours) was determined in fibronectin- or collagen-coated cell culture plate as described in “Materials and Methods.” (F) HE4 knockdown inhibited cell migration. 2008 cells were transfected with either control or HE4 siRNA and cultured until confluence. A scratch was made using a P1000 pipette tip, and images were captured at 0, 6, 12, and 18 hours (scale bar: 500 μm). Right panel: The average width of the gap (y-axis, in μm) was calculated as described in the “Materials and Methods” section.

Article Snippet: Santa Cruz Biotechnology siRNAs for control (cat. No. sc-37007) and HE4 (cat. No. sc-43826); Vectors: empty (addgene, cat. No. 24165), p65 (addgene, cat. No. 21966), IKK2-EE (addgene, cat. No.11105); antibodies: p53 (Santa Cruz Biotechnology cat. No. sc-126), HE4 (OriGene cat. No. UM870019), p50 (BioLegend cat. No. 603901), P-c-Jun (BioLegend cat. No. 605551).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Knockdown, Cell Culture, Migration, Transferring