Journal: Frontiers in Oncology

Article Title: AXL Overexpression in Tumor-Derived Endothelial Cells Promotes Vessel Metastasis in Patients With Hepatocellular Carcinoma

doi: 10.3389/fonc.2021.650963

Figure Lengend Snippet: AXL/SOX2/DKK-1 axis in HUVECs promotes HCC metastasis. (A) DKK-1 and CCL14 secretion was significantly downregulated in CM from HUVEC-AXL-KD compared with that from HUVEC-AXL-NC, as detected with a human cytokine antibody array. (B) DKK-1 and CCL14 expression was markedly upregulated in the CM of HUVECs overexpressing AXL (CCL14: p < 0.001; DKK-1: p = 0.003) compared with the CM of HUVEC-AXL-NC, as detected by ELISA assay. (C) AXL siRNA downregulated DKK-1 and CCL14 secretion in the CM of HUVEC-AXL-NC and HUVEC-AXL-OE cells (CCL14: p < 0.001 and p < 0.001; DKK-1: p < 0.001 and p < 0.001). (D) DKK1 siRNA (MHCC-97L: p < 0.001 and p < 0.001; HCC-LM3: p <0.001 and p < 0.001), but not CCL14 siRNA (MHCC-97L: p = 0.126 and p = 0.711; HCC-LM3: p = 0.901 and p = 0.694) could attenuate the effect of the CM from HUVEC-AXL-NC and HUVEC-AXL-OE cells on the migration of HCC-LM3 cells and MHCC-97L cells. (E) SOX2 mRNA expression was significantly increased in HUVEC-AXL-OE cells and decreased in HUVEC-AXL-KD cells compared with HUVEC-AXL-NC cells (HUVEC-AXL-KD: p < 0.001, HUVEC-AXL-OE: p < 0.001). (F) AXL overexpression could significantly increase SOX2 and DKK-1 protein expression in HUVEC-AXL-OE cells compared with HUVEC-AXL-NC cells, and SOX2 siRNA inhibited SOX2 and DKK-1 protein expression in HUVEC-AXL-OE and HUVEC-AXL-NC cells.

Article Snippet: The protein concentrations of CCL14 and DKK-1 in the supernatants were also measured using an enzyme-linked immunosorbent assay (ELISA) kit (CCL14: EK1123 Boster, Wuhan, China; DKK-1: EK0867 Boster, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Ab Array, Expressing, Enzyme-linked Immunosorbent Assay, Migration, Over Expression

Journal: PLoS ONE

Article Title: Wnt5a Regulates Midbrain Dopaminergic Axon Growth and Guidance

doi: 10.1371/journal.pone.0018373

Figure Lengend Snippet: Select antagonists were used to identify the downstream signaling pathway responsible for mediating the effects of Wnt5a on DA neurites. Antagonism of the canonical Wnt pathway (using Dkk1 recombinant protein), the canonical and non-canonical Wnt pathways (using casein kinase 1 inhibitor, D4476) and even more selectively the non-canonical PCP pathway (using Rac1 inhibitor, NSC23766) revealed that the effect of Wnt5a on ( A ) neurite length and ( B ) neurite number were mediated by the non-canonical PCP pathway (D4476 and NSC23766 both significantly antagonizing the effects of Wnt5a). ( C – F ) Examples of changes in DA neuron morphology following treatment with Wnt5a ± selective antagonists. Scale bar = 50 µm. Data represents mean ± s.e.m., n = 4–5 cultures; ** p<0.01, *** p<0.001.

Article Snippet: For antagonism experiments using secreted frizzled-related protein 1 (sFRP-1; 5 µg/ml, R&D Systems), Wnt5a antibody (αWnt5a; 2 µg/ml, R&D Systems), human RYK-Fc (3 µg/ml, see details below), goat anti-Frizzled3-CRD (αFz3-CRD; 3 µg/ml, R&D Systems), Dickkopf-1 (Dkk1; 500 ng/ml, R&D Systems), casein kinase 1 inhibitor (D4476, 50 mM, Roche) or Rac-1 inhibitor (NSC233766, 500 nM, Calbiochem), Wnt5a and the antagonist were added to the wells 15 minutes prior to seeding the VM cells.

Techniques: Recombinant

Journal: Pulmonary Circulation

Article Title: Shared gene expression patterns in mesenchymal progenitors derived from lung and epidermis in pulmonary arterial hypertension: identifying key pathways in pulmonary vascular disease

doi: 10.1086/688314

Figure Lengend Snippet: Reagent information

Article Snippet: Control and PAH human lung MSCs were plated at 50,000 cells per well with the following treatment conditions: untreated, 1N ammonium hydroxide (Sigma-Aldrich, St. Louis, MO, catalog no. 318612), 100 μM gallocyanine (Sigma-Aldrich, catalog no. 124508), and 100 ng/mL DKK1 (OriGene, Rockville, MD, catalog no. TP723065).

Techniques:

Journal: Pulmonary Circulation

Article Title: Shared gene expression patterns in mesenchymal progenitors derived from lung and epidermis in pulmonary arterial hypertension: identifying key pathways in pulmonary vascular disease

doi: 10.1086/688314

Figure Lengend Snippet: DKK1 regulates gene expression in isolated PAH patient BMPR2-mutant cells. A, Western blot analysis was performed with cell lysates from lung MPCs to detect DKK1 and LRP6 levels. B, C, Densitometry was performed and normalized to β-actin housekeeping levels. Three control, 2 BMPR2-mutant HPAH, and 4 IPAH patient lung MPC lines were analyzed per group. D, E, Modulation of DKK1 signaling was performed by stimulation with DKK1 treatment (100 ng/mL) or DKK1 inhibition with the antagonist gallocyanine (NCI8642; 100 μm). The resulting gene expression was analyzed after 48 hours. A qRT-PCR analysis was performed to analyze changes in gene expression by control or BMPR2-mutant HPAH lung MPCs. In addition to that of LRP6 and DKK1, expression of genes associated with pericyte differentiation (ACTA2, CSPG4) and matrix production (COL1A1, COL3A1, FN1) was also analyzed. All amplification was normalized to a housekeeping gene, and the results are presented as mean fold change over untreated controls ± standard error. *P < 0.5; **P < 0.01. F, Schematic representation of the relationship between DKK1 and LRP6. HPAH: heritable PAH; HuLung: human lung; IPAH: idiopathic PAH; MPC: mesenchymal progenitor cell; mut: mutant; PAH: pulmonary arterial hypertension; qRT-PCR: quantitative reverse transcription polymerase chain reaction; UT: untreated; Veh: gallocyanine vehicle 1N NH4OH.

Article Snippet: Control and PAH human lung MSCs were plated at 50,000 cells per well with the following treatment conditions: untreated, 1N ammonium hydroxide (Sigma-Aldrich, St. Louis, MO, catalog no. 318612), 100 μM gallocyanine (Sigma-Aldrich, catalog no. 124508), and 100 ng/mL DKK1 (OriGene, Rockville, MD, catalog no. TP723065).

Techniques: Expressing, Isolation, Mutagenesis, Western Blot, Inhibition, Quantitative RT-PCR, Amplification, Reverse Transcription Polymerase Chain Reaction

Journal: Pulmonary Circulation

Article Title: Shared gene expression patterns in mesenchymal progenitors derived from lung and epidermis in pulmonary arterial hypertension: identifying key pathways in pulmonary vascular disease

doi: 10.1086/688314

Figure Lengend Snippet: Analysis of targeted gene expression profiles of lung MPCs from COPD and IPF patients highlight that the DKK1 pathway is affected in multiple chronic pulmonary diseases. A. A qRT-PCR analysis was performed to evaluate the patterns of representative genes expressed by lung MPCs from adult chronic lung disease, including COPD and IPF, relative to control and PAH samples; n = 3 patients for each group. All amplification was normalized to a housekeeping gene, and the results presented as mean fold change over control ± standard error. *P < 0.5. B–F, Representative bright-field images of immunohistochemical localization of DKK1. Scale bar: 100 μm. G, Western blot analysis using cell lysates from lung MPC to detect DKK1 levels. Densitometry was performed and normalized to β-actin housekeeping levels. Sample: 3 control and 3 IPF MPCs. Data presented as the mean ± standard error. COPD: chronic obstructive pulmonary disease; IPF: idiopathic pulmonary fibrosis; MPCs: mesenchymal progenitor cells; PAH: pulmonary arterial hypertension; qRT-PCR: quantitative reverse transcription polymerase chain reaction.

Article Snippet: Control and PAH human lung MSCs were plated at 50,000 cells per well with the following treatment conditions: untreated, 1N ammonium hydroxide (Sigma-Aldrich, St. Louis, MO, catalog no. 318612), 100 μM gallocyanine (Sigma-Aldrich, catalog no. 124508), and 100 ng/mL DKK1 (OriGene, Rockville, MD, catalog no. TP723065).

Techniques: Expressing, Quantitative RT-PCR, Amplification, Immunohistochemical staining, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Pulmonary Circulation

Article Title: Shared gene expression patterns in mesenchymal progenitors derived from lung and epidermis in pulmonary arterial hypertension: identifying key pathways in pulmonary vascular disease

doi: 10.1086/688314

Figure Lengend Snippet: Differential gene expression between PAH and control MPCs

Article Snippet: Control and PAH human lung MSCs were plated at 50,000 cells per well with the following treatment conditions: untreated, 1N ammonium hydroxide (Sigma-Aldrich, St. Louis, MO, catalog no. 318612), 100 μM gallocyanine (Sigma-Aldrich, catalog no. 124508), and 100 ng/mL DKK1 (OriGene, Rockville, MD, catalog no. TP723065).

Techniques: Expressing

Journal: Pulmonary Circulation

Article Title: Shared gene expression patterns in mesenchymal progenitors derived from lung and epidermis in pulmonary arterial hypertension: identifying key pathways in pulmonary vascular disease

doi: 10.1086/688314

Figure Lengend Snippet: Significance of changes in gene expression in PAH MPCs versus control

Article Snippet: Control and PAH human lung MSCs were plated at 50,000 cells per well with the following treatment conditions: untreated, 1N ammonium hydroxide (Sigma-Aldrich, St. Louis, MO, catalog no. 318612), 100 μM gallocyanine (Sigma-Aldrich, catalog no. 124508), and 100 ng/mL DKK1 (OriGene, Rockville, MD, catalog no. TP723065).

Techniques: Expressing

Journal: Pulmonary Circulation

Article Title: Shared gene expression patterns in mesenchymal progenitors derived from lung and epidermis in pulmonary arterial hypertension: identifying key pathways in pulmonary vascular disease

doi: 10.1086/688314

Figure Lengend Snippet: Analysis of targeted gene expression profiles of skin mesenchymal cells from PAH BMPR2 and CAV1 mutation carriers relative to wild-type individuals (i.e., noncarriers of mutations) and unaffected mutation carriers. A, A qRT-PCR analysis was performed to validate the patterns of representative genes expressed by skin MCs; n = 3 patients for each group. The mean of combined patient samples per group and results for individual samples are presented. All amplification was normalized to a housekeeping gene, and the results are presented as mean fold change over control ± standard error. B, Representative immunofluorescent images of DKK1 and PDK4 localization in skin MCs and lung MPCs. Scale bar: 100 μm. DAPI: 4′,6-diamidino-2-phenylindole; HPAH: heritable PAH; IPAH: idiopathic PAH; MCs: mesenchymal cells; MPCs: mesenchymal progenitor cells; mut: mutant; PAH: pulmonary arterial hypertension; qRT-PCR: quantitative reverse transcription polymerase chain reaction.

Article Snippet: Control and PAH human lung MSCs were plated at 50,000 cells per well with the following treatment conditions: untreated, 1N ammonium hydroxide (Sigma-Aldrich, St. Louis, MO, catalog no. 318612), 100 μM gallocyanine (Sigma-Aldrich, catalog no. 124508), and 100 ng/mL DKK1 (OriGene, Rockville, MD, catalog no. TP723065).

Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, Amplification, Reverse Transcription Polymerase Chain Reaction

Journal: Pulmonary Circulation

Article Title: Shared gene expression patterns in mesenchymal progenitors derived from lung and epidermis in pulmonary arterial hypertension: identifying key pathways in pulmonary vascular disease

doi: 10.1086/688314

Figure Lengend Snippet: Differential gene expression between ABCG2-expressing skin and lung mesenchymal progenitor cells

Article Snippet: Control and PAH human lung MSCs were plated at 50,000 cells per well with the following treatment conditions: untreated, 1N ammonium hydroxide (Sigma-Aldrich, St. Louis, MO, catalog no. 318612), 100 μM gallocyanine (Sigma-Aldrich, catalog no. 124508), and 100 ng/mL DKK1 (OriGene, Rockville, MD, catalog no. TP723065).

Techniques: Expressing

Journal: Pulmonary Circulation

Article Title: Shared gene expression patterns in mesenchymal progenitors derived from lung and epidermis in pulmonary arterial hypertension: identifying key pathways in pulmonary vascular disease

doi: 10.1086/688314

Figure Lengend Snippet: Representative bright-field images of immunohistochemical localization of DKK1. DKK1 localization increased in the vasculature of PAH patients, independent of mutation status. Patient lung tissue was analyzed by immunostaining of paraffin-embedded lung sections with DAB detection (black). A–D, DKK1 localized to alveolar epithelium and a few endothelial cells in control sections. E–J, DKK1 was present with increasing intensity in the smooth muscle layers of PAH tissue relative to control. Sample: 3 controls, 3 HPAH patients, 3 IPAH patients. Scale bar: 100 μm. DAB: diaminobenzidine; HPAH: heritable PAH; IPAH: idiopathic PAH; mut: mutant; PAH: pulmonary arterial hypertension.

Article Snippet: Control and PAH human lung MSCs were plated at 50,000 cells per well with the following treatment conditions: untreated, 1N ammonium hydroxide (Sigma-Aldrich, St. Louis, MO, catalog no. 318612), 100 μM gallocyanine (Sigma-Aldrich, catalog no. 124508), and 100 ng/mL DKK1 (OriGene, Rockville, MD, catalog no. TP723065).

Techniques: Immunohistochemical staining, Mutagenesis, Immunostaining

Journal: Frontiers in Oncology

Article Title: Chondroitin Sulfates Control Invasiveness of the Basal-Like Breast Cancer Cell Line MDA-MB-231 Through ROR1

doi: 10.3389/fonc.2022.914838

Figure Lengend Snippet: CS-E-elicited invasiveness is enhanced by the absence of DKK1. (A) Raw sensor grams. ROR1 was immobilized in a flow cell of a CM5 sensor chip. DKK1 alone (a) , DKK1 premixed with CS-E at a 1:7 molar ratio (b) , and DKK1 premixed with CS-A at a 1:7 molar ration (c) were used as analytes. (B) Response-unit quantification of binding. (C) DKK1 mRNA expression in MDA-MB-231 cells transfected with siDKK1 or control siRNA ( siCont ) measured using qPCR (n=4). Expression data were normalized to those of GAPDH . (D) Invasiveness of DKK1 knocked down MDA-MB-231 cells ( siDKK1 ) or control cells ( siCont ) treated with or without CS-E (n>5). Data were analyzed using a Tukey–Kramer multiple comparison.

Article Snippet: For ROR1 binding assays, WNT5A (0, 0.038, 0.075, 0.15, 0.30, and 0.60 μM), WNT5A/CS-E (0, 0.036/0.25, 0.071/0.50, 0.15/1.0, 0.29/2.0, and 0.57/4.0 μM), recombinant human DKK1 (Cat. No. 5439-DK/CF, R&D Systems) (0, 0.038, 0.075, 0.15, and 0.3 μM), or DKK1/CS-E (0, 0.038/0.28, 0.075/0.55, 0.15/1.1, and 0.3/2.1 μM) were sequentially injected at a flowrate of 30 μl/min for 120 s at 25°C; the dissociation time was set for 130 s. Binding reactions were performed in 50 mM Tris–HCl buffer (pH 7.5).

Techniques: Binding Assay, Expressing, Transfection, Control, Comparison

Journal: Frontiers in Oncology

Article Title: Chondroitin Sulfates Control Invasiveness of the Basal-Like Breast Cancer Cell Line MDA-MB-231 Through ROR1

doi: 10.3389/fonc.2022.914838

Figure Lengend Snippet: Schematic of CS-E enhancement of invasive activity of the triple-negative breast cancer MDA-MB-231 cell line. CS chains bind WNT5A and ROR1 through E units, signaling cancer cells to activate JNK1. Decreasing E units by knockdown of CHST11 and CHST15 inhibits WNT5A−ROR1−JNK signaling. DKK1 suppresses CS tumor promoting activity by binding to E units.

Article Snippet: For ROR1 binding assays, WNT5A (0, 0.038, 0.075, 0.15, 0.30, and 0.60 μM), WNT5A/CS-E (0, 0.036/0.25, 0.071/0.50, 0.15/1.0, 0.29/2.0, and 0.57/4.0 μM), recombinant human DKK1 (Cat. No. 5439-DK/CF, R&D Systems) (0, 0.038, 0.075, 0.15, and 0.3 μM), or DKK1/CS-E (0, 0.038/0.28, 0.075/0.55, 0.15/1.1, and 0.3/2.1 μM) were sequentially injected at a flowrate of 30 μl/min for 120 s at 25°C; the dissociation time was set for 130 s. Binding reactions were performed in 50 mM Tris–HCl buffer (pH 7.5).

Techniques: Activity Assay, Knockdown, Binding Assay