Journal: Biochemistry and Biophysics Reports

Article Title: Hepatoma-derived growth factor and non-coding RNA network in ovarian cancer patients

doi: 10.1016/j.bbrep.2025.102168

Figure Lengend Snippet: ROC of CA125, HE4 and, HDGF.

Article Snippet: Enzyme-linked immunosorbent assays (ELISAs) were performed to measure serum levels of HDGF (BT Laboratories, China), HE4 (Xema, Russia), and CA125 (Pishtaz-Teb, Iran) using commercially available kits, following the manufacturers’ protocols.

Techniques:

Journal: medRxiv

Article Title: The founder missense mutation of WFDC2 in Koreans leads to severe respiratory distress accompanied by bronchiectasis and rhinosinusitis

doi: 10.1101/2024.11.16.24317083

Figure Lengend Snippet: (A, B) Summary and result of WES/WGS analysis to elucidate the causal variants in patients with severe bronchiectasis and chronic rhinosinusitis. Five families had a homozygous variant in WFDC2 , which was the c.291C>G substitution resulting in the amino acid change p.Cys97Trp. (C) Pedigree of five families with the c.291C>G WFDC2 variant. The probands are indicated using arrows. (D) Sanger sequencing result of c.291C>G WFDC2 variant representing individuals from three families. The altered amino acid is written in red. WES, Whole exome sequencing; WGS, whole genome sequencing.

Article Snippet: Plasmids encoding human WFDC2 (NM_006103.4) and mouse Wfdc2 (NM_026323.2, isoform 1 and NM_001374655.1, isoform 2) were purchased from OriGene (Rockville, MD, USA).

Techniques: Variant Assay, Sequencing

Journal: medRxiv

Article Title: The founder missense mutation of WFDC2 in Koreans leads to severe respiratory distress accompanied by bronchiectasis and rhinosinusitis

doi: 10.1101/2024.11.16.24317083

Figure Lengend Snippet: (A) Overall structure of the WFDC2 protein. Two WAP domains and the disulfide bond-forming cysteines are shown. C97 forms a disulfide bond with C109. (B) Multiple sequence alignment of human WFDC2 against its orthologs from five different vertebrate species was performed using Jalview. The mutated amino acid residue (C97) and the disulfide bond-forming cysteine residue with C97 (C109) are indicated. (C) Monomer and homo-dimer structure prediction of WFDC2 WT and p.C97W mutant by AlphaFold2. , (D) Western blot analysis of WT and p.C97W mutant WFDC2 proteins in both the lysates and secreted media of HEK293T cells overexpressing the proteins. In (D), data are presented in mean ± standard error of the mean. In (D), a two-way Student’s t-test was used to calculate the p-value, and a p-value < 0.05 was considered significant. *, p < 0.05; ****, p < 0.0001.

Article Snippet: Plasmids encoding human WFDC2 (NM_006103.4) and mouse Wfdc2 (NM_026323.2, isoform 1 and NM_001374655.1, isoform 2) were purchased from OriGene (Rockville, MD, USA).

Techniques: Sequencing, Residue, Mutagenesis, Western Blot

Journal: medRxiv

Article Title: The founder missense mutation of WFDC2 in Koreans leads to severe respiratory distress accompanied by bronchiectasis and rhinosinusitis

doi: 10.1101/2024.11.16.24317083

Figure Lengend Snippet: (A) Overall structure of the WFDC2 protein. Two WAP domains and the disulfide bond-forming cysteines are shown. C97 forms a disulfide bond with C109. (B) Multiple sequence alignment of human WFDC2 against its orthologs from five different vertebrate species was performed using Jalview. The mutated amino acid residue (C97) and the disulfide bond-forming cysteine residue with C97 (C109) are indicated. (C) Monomer and homo-dimer structure prediction of WFDC2 WT and p.C97W mutant by AlphaFold2. , (D) Western blot analysis of WT and p.C97W mutant WFDC2 proteins in both the lysates and secreted media of HEK293T cells overexpressing the proteins. In (D), data are presented in mean ± standard error of the mean. In (D), a two-way Student’s t-test was used to calculate the p-value, and a p-value < 0.05 was considered significant. *, p < 0.05; ****, p < 0.0001.

Article Snippet: Plasmids encoding human WFDC2 (NM_006103.4) and mouse Wfdc2 (NM_026323.2, isoform 1 and NM_001374655.1, isoform 2) were purchased from OriGene (Rockville, MD, USA).

Techniques: Sequencing, Residue, Mutagenesis, Western Blot

Journal: Scientific reports

Article Title: HE4 expression is associated with hormonal elements and mediated by importin-dependent nuclear translocation.

doi: 10.1038/srep05500

Figure Lengend Snippet: Figure 1 | HE4 overexpression confers resistance against antiestrogens in epithelial ovarian cancer cells, and estrogen/antiestrogens upregulate HE4 expression. (a–b) MTS cell viability assays for SKOV3 wild type (WT), null vector (NV), and stably HE4-overexpressing clone-1 (C1). Cells were treated overnight in serum-deprived media with various concentrations of fulvestrant (a) or tamoxifen (b) in triplicate. Fulvestrant and tamoxifen had little to no effect on SKOV3 C1 at concentrations that were cytotoxic to SKOV3 WT and NV. (c–d) Confocal images of fixed SKOV3 WT and OVCAR8 WT cells stained with HE4 primary antibody and corresponding DyLight fluorescent secondary antibody and chromatin staining (DAPI). Cells were stimulated with estradiol, tamoxifen, or fulvestrant overnight in serum-deprived media. Each of these resulted in increased nuclear or nucleolar HE4 expression relative to unstimulated cells.

Article Snippet: Cells were then stained for HE4 primary antibody (TA307787, Origene, Rockville, MD, USA) at 15500, followed by Dylight 595 secondary antibody (151500).

Techniques: Over Expression, Expressing, Plasmid Preparation, Stable Transfection, Staining

Journal: Scientific reports

Article Title: HE4 expression is associated with hormonal elements and mediated by importin-dependent nuclear translocation.

doi: 10.1038/srep05500

Figure Lengend Snippet: Figure 2 | Estrogen receptor-a (ER-a) expression is abrogated in HE4-overexpressing SKOV3 C1, and HE4 interacts with ER-a. (a) Cellular lysates of SKOV3 WT, NV, and C1 grown to 80% confluency in serum-supplemented media were probed for ER-a. SKOV3 C1 demonstrated almost complete absence of ER-a expression, in comparison to SKOV3 WT and NV. (b) Confocal images of fixed SKOV3 NV and C1 cells stained ER-a primary antibody. Cells were either untreated or stimulated with estradiol or fulvestrant overnight in serum-deprived media. In SKOV3 NV, ER-a degraded and internalized to the nucleus upon stimulation with estradiol or fulvestrant, but ER-a signaling was absent in SKOV3 C1. (c) HE4 was immunoprecipitated from lysates of SKOV3 WT, NV, and C1 cells with isotype-matched IgG as a control, and western blot was performed. ER-a co-immunoprecipitation with HE4 was detected in SKOV3 WT and NV, and was not present in SKOV3 C1. (d) Paraffin-embedded ovarian tissue samples from human patients were categorized based on HE4 serum levels and histopathological diagnosis. Tissues were deparaffinized and stained with ER-a primary antibody and examined with confocal microscopy. Serous and endometrioid HE4 low-expressing tumor tissues exhibited strong nuclear ER-a staining in epithelial cells. Meanwhile, serous HE4 high-expressing tissue demonstrated reduced ER-a staining in epithelial cell nuclei.

Article Snippet: Cells were then stained for HE4 primary antibody (TA307787, Origene, Rockville, MD, USA) at 15500, followed by Dylight 595 secondary antibody (151500).

Techniques: Expressing, Comparison, Staining, Immunoprecipitation, Control, Western Blot, Biomarker Discovery, Confocal Microscopy

Journal: Scientific reports

Article Title: HE4 expression is associated with hormonal elements and mediated by importin-dependent nuclear translocation.

doi: 10.1038/srep05500

Figure Lengend Snippet: Figure 3 | Stable HE4 overexpression in SKOV3 C1 leads to reduced 5-methylcytosine activity. (a) Confocal images of untreated SKOV3 WT, NV, and C1 cells stained for nuclear expression of 5-methylcytosine (5 MC). Nuclear 5 MC expression was observed in SKOV3 WT and NV, but was largely absent in HE4-overexpressing SKOV3 C1. (b) SKOV3 NV and C1 were stimulated with exogenous recombinant HE4 in serum-deprived media overnight, and stained for 5 MC. Recombinant HE4 resulted in increased 5 MC activity in SKOV3 NV, but did not impact 5 MC expression in SKOV3 C1 cells with preexisting cellular machinery for HE4 overexpression. (c) Paraffin-embedded ovarian human tissue samples were categorized based on HE4 serum levels and histopathological diagnosis, and stained with 5 MC primary antibody. Nuclear 5 MC activity was present in normal ovarian tissue and benign serous tumor tissue, but this nuclear expression was abrogated in HE4 high-expressing serous and endometrioid tumor tissue.

Article Snippet: Cells were then stained for HE4 primary antibody (TA307787, Origene, Rockville, MD, USA) at 15500, followed by Dylight 595 secondary antibody (151500).

Techniques: Over Expression, Activity Assay, Staining, Expressing, Recombinant, Biomarker Discovery

Journal: Scientific reports

Article Title: HE4 expression is associated with hormonal elements and mediated by importin-dependent nuclear translocation.

doi: 10.1038/srep05500

Figure Lengend Snippet: Figure 4 | Importin-4 interacts with HE4 to facilitate nuclear transport. (a) Confocal microscopy of unstimulated SKOV3 WT cells in serum- supplemented media, stained for HE4 (green fluorescence) and importin-4 (red fluorescence). Strong colocalization (yellow-colored regions) was observed between HE4 and importin-4. (b) Co-immunoprecipitation was conducted using lysates from untreated SKOV3 WT, NV, and C1 cells, with HE4 as the primary bait antibody and isotype-matched IgG as a control. In a second set of lysates, importin-4 was used as bait. Western blot was performed and probed for importin-4 primary antibody. An increase in importin-4 co-immunoprecipitation was detected in HE4-overexpressing SKOV3 C1, compared to WT and NV (blot is cropped for image clarity, uncropped image is pictured in supplementary material). (c) Confocal microscopy of unstimulated SKOV3 C1 cells in serum-supplemented media, stained for HE4 (red fluorescence) and importin-4 (green fluorescence). In addition to strong colocalization between HE4 and importin-4, basal levels of nuclear HE4 expression were increased relative to SKOV3 WT.

Article Snippet: Cells were then stained for HE4 primary antibody (TA307787, Origene, Rockville, MD, USA) at 15500, followed by Dylight 595 secondary antibody (151500).

Techniques: Confocal Microscopy, Staining, Fluorescence, Immunoprecipitation, Control, Western Blot, Expressing

Journal: Scientific reports

Article Title: HE4 expression is associated with hormonal elements and mediated by importin-dependent nuclear translocation.

doi: 10.1038/srep05500

Figure Lengend Snippet: Figure 5 | Ivermectin, an importin inhibitor, inhibits HE4 nuclear translocation and sensitizes HE4-overexpressing SKOV3 C1 to fulvestrant and tamoxifen treatment. (a–b) Confocal microscopy of SKOV3 WT and OVCAR8 WT cells stained with HE4 primary antibody. Cells were pre-treated ivermectin, followed by stimulation with estradiol or fulvestrant and co-administration of ivermectin in serum-free media overnight. Ivermectin treatment inhibited nuclear and nucleolar HE4 translocation typically induced by estradiol and fulvestrant in both SKOV3 and OVCAR8 WT. (c–d) MTS cell viability assay was performed in triplicate in SKOV3 WT, NV, and C1. Cells were either untreated or pretreated with ivermectin, followed by overnight treatment in serum-free media with various concentrations of fulvestrant and tamoxifen. Cells that received ivermectin pretreatment were also co- administered ivermectin along with the fulvestrant or tamoxifen. Ivermectin treatment sensitized SKOV3 C1 to concentrations of fulvestrant and tamoxifen that were non-cytotoxic without ivermectin. Furthermore, the viability levels of SKOV3 C1 cells sensitized by ivermectin to fulvestrant and tamoxifen were comparable to those of SKOV3 NV.

Article Snippet: Cells were then stained for HE4 primary antibody (TA307787, Origene, Rockville, MD, USA) at 15500, followed by Dylight 595 secondary antibody (151500).

Techniques: Translocation Assay, Confocal Microscopy, Staining, Viability Assay

Journal: Frontiers in Pharmacology

Article Title: Human Epididymis Secretory Protein 4 (HE4) Compromises Cytotoxic Mononuclear Cells via Inducing Dual Specificity Phosphatase 6

doi: 10.3389/fphar.2019.00216

Figure Lengend Snippet: Genes induced in response to HE4.

Article Snippet: Phenotypes of the clones were evaluated by western blotting using anti-HE4 antibody (Origene, TA801137; ).

Techniques: Variant Assay

Journal: Frontiers in Pharmacology

Article Title: Human Epididymis Secretory Protein 4 (HE4) Compromises Cytotoxic Mononuclear Cells via Inducing Dual Specificity Phosphatase 6

doi: 10.3389/fphar.2019.00216

Figure Lengend Snippet: HE4 upregulates expression of DUSP6 in PBMCs. (A) DUSP6 transcription in response to a 6-hr incubation with 0.01 mg/mL rHE4 (HE4) or equivalent amount of PBS (CTR) were evaluated by triplicated trials of real time PCR using PBMCs from four individual donors. (B) Two-color flow cytometric analysis of PBMC following 24-h incubation with 0.01 mg/mL of rHE4 (HE4) or vehicle (CTR). 2D-scatterplots of DUSP6 (Alexa Fluor 647) and CD3 or CD56 (FITC) are shown. Numbers on the plots indicate mean percentages of the cell populations of the each tetrameric area from four independent experiments. (C) Bar graphs from flow cytometric analyses using PBMCs from four individual donors. The mean ± SEM are shown. * p < 0.05, ** p < 0.01.

Article Snippet: Phenotypes of the clones were evaluated by western blotting using anti-HE4 antibody (Origene, TA801137; ).

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction

Journal: Frontiers in Pharmacology

Article Title: Human Epididymis Secretory Protein 4 (HE4) Compromises Cytotoxic Mononuclear Cells via Inducing Dual Specificity Phosphatase 6

doi: 10.3389/fphar.2019.00216

Figure Lengend Snippet: DUSP6 concenrations in cell lysates and culture media of PBMC.

Article Snippet: Phenotypes of the clones were evaluated by western blotting using anti-HE4 antibody (Origene, TA801137; ).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Human Epididymis Secretory Protein 4 (HE4) Compromises Cytotoxic Mononuclear Cells via Inducing Dual Specificity Phosphatase 6

doi: 10.3389/fphar.2019.00216

Figure Lengend Snippet: HE4 upregulates expression of DUSP6 in peripheral CD8 + T cells. Two-color flow cytometric analysis of PBMCs following 24-h incubation with 0.01 mg/mL of rHE4 (HE4) or vehicle (CTR). (A) 2D-scatterplots of DUSP6 (Alexa Fluor 647) and CD4 or CD8 (FITC) are shown. Numbers on the plots indicate mean percentages of the cell populations of the each tetrameric area from four independent experiments. (B) A bar graph from flow cytometric analyses using PBMCs from four individual donors. The mean using ± SEM are shown. * p < 0.01.

Article Snippet: Phenotypes of the clones were evaluated by western blotting using anti-HE4 antibody (Origene, TA801137; ).

Techniques: Expressing, Incubation

Journal: Frontiers in Pharmacology

Article Title: Human Epididymis Secretory Protein 4 (HE4) Compromises Cytotoxic Mononuclear Cells via Inducing Dual Specificity Phosphatase 6

doi: 10.3389/fphar.2019.00216

Figure Lengend Snippet: HE4 suppresses Erk1/2 phosphorylation in CD8 + and CD56 + cells via DUSP6 induction. Two-color flow cytometric analysis of PBMC following 24-hr incubation with PBS (CTR), rHE4 (0.01 mg /mL) and BCI (1 mM) as indicated. (A) 2-D scatterplots of phosphor-Erk1/2 (Alexa Fluor 647) and CD8 or CD56 (FITC) are shown. (B) Mean ± SEM from analyses of phosphor-Erk1/2 positive cells from four individual donors are shown in the bar graph. (C) Immunoblotting for phosphor-Erk1/2 in CD56 + NK92MI, CD8 + TALL-104 and CD4 + H9 cells following 1-h incubation with the conditioned media from 24-h PBMC culture with rHE4 (0.01 mg/mL) and BCI (1 mM) in the indicated combinations. Blots of total Erk1/2 are shown as loading controls. (D) Bar graph represents the relative band densities to controls. Mean ± SEM are shown ( n = 4). (E) DUSP6 concentrations in cell lysates and culture supernatants (SN) after 24 h incubation with or without rHE4 (0.01 mg/mL). Mean ± SEM are shown ( n = 4). * p < 0.05, ** p < 0.01, n.s., not significant.

Article Snippet: Phenotypes of the clones were evaluated by western blotting using anti-HE4 antibody (Origene, TA801137; ).

Techniques: Phospho-proteomics, Incubation, Western Blot

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 HE4 in ovarian cancer cell lines. mRNA expression of HE4 was determined by semiquantitative reverse transcription PCR. GAPDH served as a loading control. NTC: Non-template control. (B) TNFα and IL-1β promote the secretion of HE4 in ovarian cancer cell lines. Cells were subjected to TNFα (30 ng/mL) or IL-1β (10 ng/mL) treatment for 72 hours. Afterward, cell culture medium was collected. HE4 levels 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, Reverse Transcription, Control, Cell Culture, Enzyme-linked Immunosorbent 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: 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, 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) 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, 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