Journal:

Article Title: Mitochondrial Dysfunction and Reactive Oxygen Species Imbalance Promote Breast Cancer Cell Motility through a CXCL 14-Mediated Mechanism

doi: 10.1158/0008-5472.CAN-08-3359

Figure Lengend Snippet: CXCL14 promotes cell motility. A, analysis of CXCL14 expression in MCF7 cells and subclones by RT-PCR (top) and by Western blotting (bottom). B, addition of exogenous CXCL14 promoted MCF7 cell migration in vitro . Plated MCF7 cells were grown to confluency, wounded, and then incubated with either 0.57 ng CXCL14 or conditioned medium from H or P clones for 48 h. Each plate was examined by phase-contrast microscopy for the amount of wound closure. C, transient transfection of CXCL14 promotes MCF7 cell migration in vitro . MCF7 cells were transfected with 0.5 µg of CXCL14 plasmid. After 48 h, cells were collected and used in a migration assay. D, inhibition of invasion of clone H by AP-1 decoy. Clone H cells were transfected with either AP-1decoy or scramble decoy (100 pmol). After 48 h, cells were collected and lysed and CXCL14 expression was analyzed by Western blotting (left ), or cells were collected and cultured on transwell cell culture inserts coated with Matrigel for an additional 20 h (right). Results of a representative experiment (n = 3).

Article Snippet: Human CXCL14 cDNA clone was from Origene Technologies, Inc.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Migration, In Vitro, Incubation, Clone Assay, Microscopy, Transfection, Plasmid Preparation, Inhibition, Cell Culture

Journal:

Article Title: Mitochondrial Dysfunction and Reactive Oxygen Species Imbalance Promote Breast Cancer Cell Motility through a CXCL 14-Mediated Mechanism

doi: 10.1158/0008-5472.CAN-08-3359

Figure Lengend Snippet: ROS promotes CXCL14 expression in MCF7 subclone cells through activation of AP-1. A, ROS-promoted CXCL14 expression. The induction of CXCL14 mRNA expression by H2O2 is time and dose dependent (top). MCF7 cells were treated with 20 or 100 µmol/L H2O2. RNA was isolated at the indicated time, and CXCL14 mRNA expression was analyzed. NAC down-regulated the mRNA expression of CXCL14 in clone P (bottom). Clone P cells were treated with 5 mmol/L NAC. RNA was isolated at the indicated time, and CXCL14 mRNA expression was analyzed. B, DNA binding activity of AP-1 in MCF7 cells and its subclones (left ) and H2O2-activated DNA binding activity of AP-1in MCF7 cells (right ). Nuclear extract from MCF7 cells was prepared and incubated with H2O2 at the indicated concentration for 20 min at 4°C, and then the DNA binding activity of AP-1was determined. C, effects of JNK inhibitor on the DNA binding activity of AP-1in MCF7 subclones (left ). Effects of JNK inhibitor on CXCL14 mRNA expression (right top) and protein expression (right bottom) in clone H. D, detection of the in vivo binding of AP-1 protein to the human CXCL14 promoter in a chromatin immunoprecipitation assay. Results of a representative experiment (n = 3).

Article Snippet: Human CXCL14 cDNA clone was from Origene Technologies, Inc.

Techniques: Expressing, Activation Assay, Isolation, Binding Assay, Activity Assay, Incubation, Concentration Assay, In Vivo, Chromatin Immunoprecipitation

Journal:

Article Title: Mitochondrial Dysfunction and Reactive Oxygen Species Imbalance Promote Breast Cancer Cell Motility through a CXCL 14-Mediated Mechanism

doi: 10.1158/0008-5472.CAN-08-3359

Figure Lengend Snippet: CXCL14 induced Ca2+ release from ER stores through IP3R. A, CXCL14 was localized in the ER. Western blot analysis of CXCL14 from subcellular fractions of MCF7 cells and its subclones. B, MCF7 subclones exhibited higher cytosolic Ca2+ content, as measured with Calcium Green (left ). ER Ca2+ content was decreased in MCF7 subclones, as measured with ER-Tracker Blue-White DPX (right ). *, P < 0.05, compared with the parental MCF7 cells. C, increase of cytosolic Ca2+ by CXCL14 (0.75 µg) in MCF7 cells 48 h posttransfection (left ). The cytosolic Ca2+ content increased after CXCL14 transfection in MCF7 in a dose-dependent manner (right ). D, coimmunoprecipitation of CXCL14 with IP3R. Results of a representative experiment (n = 3).

Article Snippet: Human CXCL14 cDNA clone was from Origene Technologies, Inc.

Techniques: Western Blot, Transfection

Journal:

Article Title: Mitochondrial Dysfunction and Reactive Oxygen Species Imbalance Promote Breast Cancer Cell Motility through a CXCL 14-Mediated Mechanism

doi: 10.1158/0008-5472.CAN-08-3359

Figure Lengend Snippet: CXCL14-induced Ca2+ release in microsomes and its inhibition by 2-APB in clone P in vitro. A, CXCL14 prevented the inhibition of IP3R by 2-APB in MCF7 subclones. Cells were loaded with 5 µmol/L Fluo-3 for 1h. MCF7 (red curve) and clone P (green curve) were treated with 2 µmol/L thapsigargin (TG) to deplete intracellular stores and activate capacitative calcium entry. MCF7 (brown curve) and clone P (blue curve) were treated with 100 µmol/L 2-APB for 10 min before adding 2 µmol/L thapsigargin. The results of a representative recording of the free Ca2+ measured by Fluo-3 fluorescence using flow cytometry as described in Materials and Methods are shown (n = 3). B, CXCL14 induced Ca2+ release from microsomes in a time-dependent manner. Microsomes were incubated with 1 µg CXCL14 protein, and at the indicated time, they were spun down and the supernatant containing Ca2+ was kept to further evaluate Ca2+ released. C, CXCL14 induced Ca2+ release from microsomes in a dose-dependent manner. Microsomes were incubated with up to 1 µg CXCL14 protein for 2 h and then were spun down and the supernatant was used to evaluate Ca2+ released. D, comparison of Ca2+ released from microsomes induced by IP3, thapsigargin, or CXCL14. Microsomes were incubated with 0.5 or 1 µg of CXCL14 protein, 10 µmol/L IP3, or 1 0µmol/L thapsigargin for 1 h and then spun down, and the supernatant was used to evaluate Ca2+ released. Columns, mean of at least three independent experiments; bars, SD.

Article Snippet: Human CXCL14 cDNA clone was from Origene Technologies, Inc.

Techniques: Inhibition, In Vitro, Fluorescence, Flow Cytometry, Incubation

Journal: PLoS ONE

Article Title: Regulation of epithelial migration by epithelial cell adhesion molecule requires its Claudin-7 interaction domain

doi: 10.1371/journal.pone.0204957

Figure Lengend Snippet: (A) SDS extracts from MDCK cells Mhe16 and Mhe33 lines were immunoblotted for EpCAM and GAPDH. MDCK cells express endogenous EpCAM (ME) whereas MhE lines express both ME and untagged human EpCAM (HE). Graph shows quantification of EpCAM protein levels normalized to GAPDH protein levels in the same samples. For total EpCAM protein levels in Mhe16 and MhE33, intensities for MDCK-endogenous EpCAM (ME) and human EpCAM (HE) were combined. Error bars: S.E.M. of three samples for each cell line. Compared to parental MDCK cells, the EpCAM level was 11x higher in MhE16 and 10x higher in MhE33 cells. ( B) Phase images (top) and quantification of area/cell (graph) of individual colonies of indicated cell lines. Scale bars: 20 μm. Area per cell was significantly decreased in MhE16 and MhE33 cells with *** p values < 0.0001 (two-tailed Mann-Whitney) compared to MDCK cells. Error bars: S.E.M. of 90 MDCK, 268 MhE16 and 284 MhE33 small colonies of 5 to 200 cells. ( C) Phase images of indicated cell lines at 0 hrs migration (top row) and at 6 hrs migration (middle row). The bottom row shows an overlay of “6 hrs” images onto “0 hrs” images. Scale bars: 100 μm. Graph in (C) shows quantification of average change of area/image after 6 hrs migration for each cell line. Bars: S.E.M. of nine (MDCK), four (MhE16) or five (MhE33) experiments. MDCK values are the same as shown in the graph of . Stars correspond to p values derived from unpaired Student’s t -test. For MhE16 ** p = 0.0063 and for MhE33 ** p = 0.0074 compared to MDCK.

Article Snippet: For the generation of MDCK cell lines MhE16 and MhE33 overexpressing EpCAM, EpCAM expression vector SC322331 was obtained from OriGene Technologies, MD.

Techniques: Two Tailed Test, MANN-WHITNEY, Migration, Derivative Assay

Journal: PLoS ONE

Article Title: Regulation of epithelial migration by epithelial cell adhesion molecule requires its Claudin-7 interaction domain

doi: 10.1371/journal.pone.0204957

Figure Lengend Snippet: (A) Wildtype MDCK, and MDCK lines overexpressing human EpCAM (MhE16, MhE33) were SDS extracted one day after plating at subconfluent cell density; levels of ERK and phospho-ERK were analyzed in the same immunoblot. The graph shows quantification of combined 44 and 42 kDa phospho-ERK protein levels normalized to combined 44 and 42 kDa ERK protein levels in the same sample. Error bars: S.E.M. of three independent samples for each cell line; p values derived from unpaired Student’s t test: * p = 0.023 for MhE16 to MDCK and *** p = 0.0001 for MhE33 to MDCK. (B) Phospho-ERK levels in MDCK cells, control shRNA-expressing MDCK cells (ctrl sh) and EpCAM-depleted MDCK cells (Esh2) were analyzed as in (A). Error bars: S.E.M. of six independent samples for each cell line; p values derived from unpaired Student’s t test: ** p = 0.0013 for Esh2 to MDCK and *** p = 0.0003 for Esh2 to ctrl sh.

Article Snippet: For the generation of MDCK cell lines MhE16 and MhE33 overexpressing EpCAM, EpCAM expression vector SC322331 was obtained from OriGene Technologies, MD.

Techniques: Western Blot, Derivative Assay, Control, shRNA, Expressing

Journal: PLoS ONE

Article Title: Regulation of epithelial migration by epithelial cell adhesion molecule requires its Claudin-7 interaction domain

doi: 10.1371/journal.pone.0204957

Figure Lengend Snippet: (A) EpCAM-depleted MDCK lines Esh1, Esh2 and control shRNA line ctrl sh were SDS extracted one day after plating at subconfluent cell density. Protein levels of Claudin-7 and of GAPDH as a loading control were analyzed in the same immunoblot. The graphs show quantification of Claudin-7 protein normalized to GAPDH levels in the same sample. Error bars: S.E.M. of four samples for each cell line; p values derived from unpaired Student’s t test: ** p = 0.0042 for Esh1 to ctrl sh and ** p = 0.0057 for Esh2 to ctrl sh. (B) Claudin-7 protein levels in MDCK cells and cell lines MhE16, MhE33 overexpressing human EpCAM were analyzed as described in (A). Arbitrary units for protein intensities in Y-axis (AU) x10 3 ; error bars: S.E.M. of three samples for each cell line; p value derived from unpaired Student’s t test: * p = 0.042 for MhE16 to MDCK. (C) Confocal images were acquired of cells 24 hours after plating on IBIDI chambers and 0 hours after removal of the stencil. The figure shows maximum intensity projections and orthogonal projections of confocal stacks of MDCK, MhE16 and Esh2 cells stained for nuclei (blue), EpCAM (green), and Claudin-7 (red). In the MDCK and MhE16 lines, both EpCAM and Claudin-7 localize along basolateral membrane. In the Esh2 line, Claudin-7 does not distribute throughout the basolateral membrane, and EpCAM staining is very weak. In Esh2 line, greater contrast was applied post-processing for clarity. Nuclear signal in EpCAM channel in Esh2 is due to residual mCherry signal in the nucleus post methanol fixation. Scale bar is 10μm.

Article Snippet: For the generation of MDCK cell lines MhE16 and MhE33 overexpressing EpCAM, EpCAM expression vector SC322331 was obtained from OriGene Technologies, MD.

Techniques: Control, shRNA, Western Blot, Derivative Assay, Staining, Membrane

Journal: International journal of biological sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway.

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: Figure 1. CXCL14 is overexpressed in lung cancer. (A) Difference in CXCL14 expression between normal and lung adenocarcinoma (LUAD) tissues was analyzed by meta-analysis using data downloaded from Lung Cancer Explorer (LCE). (B-D) The gene expression of CXCL14 in normal lung and LUAD obtained from The Cancer Genome Atlas (TCGA). (E, F) CXCL14 mRNA expression in NSCLC and normal lung tissues in GSE18842 and GSE11969 from the Gene Expression Omnibus (GEO) database. (G) Expression of CXCL14 in normal and tumor lung tissues stained with immunohistochemistry (IHC) and different stages was analyzed using the H score (n = 8). Scale bar = 200 µm. (H) The expression of the CXCL14 protein in MRC-5, H1299, and A549 cells was examined using Western blot (n = 4). (I) CXCL14 secretion was examined in MRC-5, H1299, and A549 cells using an enzyme-linked immunosorbent assay (ELISA; n = 4). Normal tissues and MRC-5 cells were used as controls.

Article Snippet: Lentivirus transduction and generation of stable cell lines CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Expressing, Gene Expression, Staining, Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: International journal of biological sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway.

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: Figure 2. CXCL14 promotes migration and regulates the epithelial mesenchymal transition in lung cancer cells. (A, B) H1299 cells were treated with CXCL14 (1–30 ng/ml) for 24 h, cell migration was measured with migration and wound healing assays (n = 4). (C) H1299 and A549 cells were treated with CXCL14 (1–30 ng/ml) for 24 h, cell viability was evaluated using a CCK-8 assay (n = 4). (D) Expression of CDH2 mRNA in NSCLC and normal lung tissue in GSE18842 from the GEO database. (E) Correlation analysis of CXCL14 and CDH2 in GSE18842. (F) After treatment with CXCL14 (30 ng/ml) for 24 h, the expression levels of E-cadherin, ZO-1, N-cadherin and vimentin were determined in H1299 and A549 cells on immunofluorescence (IF)-stained slides. Scale bar = 20 µm. (G, H) After incubation with CXCL14 (1–30 ng/ml) for 24 h, the expression of epithelial mesenchymal transition (EMT) markers in the H1299 and A549 cells were examined by Western blot (n = 4). Untreated cells were used as controls.

Article Snippet: Lentivirus transduction and generation of stable cell lines CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Migration, CCK-8 Assay, Expressing, Immunofluorescence, Staining, Incubation, Western Blot

Journal: International journal of biological sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway.

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: Figure 3. ACKR2 is overexpressed and regulates migration in lung cancer. (A-C) Differences in ACKR2, CXCR4, and GPR85 expression between normal and lung adenocarcinoma (LUAD) tissues were determined through meta-analysis. (D) The expression levels of ACKR2 in immunohistochemistry (IHC)-stained normal and different stage of tumor lung tissues were analyzed using the H score (n = 8). Scale bar = 200 µm. (E) The expression of the ACKR2 protein in MRC-5, H1299 and A549 cells was examined by Western blot. Data are shown as fold of MRC-5 (n = 4). (F, H) H1299 cells were transfected with control siRNA or specific ACKR2, CXCR4, and GPR85 siRNA for 24 h and incubated with CXCL14 (30 ng/ml) for another 24 h (n = 4). Cell migration was assessed through migration and wound healing assays. (G, I) H1299 cells were incubated with IgG or ACKR2, CXCR4, and GPR85 neutralized antibodies (1 µg/ml) for 1 h and incubated with CXCL14 (30 ng/ml) for another 24 h (n = 4). Cell migration was assessed through migration and wound healing assays. Normal tissues, MRC-5 cells, cells transfected with control siRNA, and incubated with IgG were used as controls.

Article Snippet: Lentivirus transduction and generation of stable cell lines CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Migration, Expressing, Immunohistochemistry, Staining, Western Blot, Transfection, Control, Incubation

Journal: International journal of biological sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway.

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: Figure 4. CXCL14 promotes cell migration and EMT marker expression via ACKR2 and PLCβ3, PKCα and c-Src in lung cancer cells. (A) A549 cells were pretreated with U73122 (3 µM), GF109203X (1 µM) or PP2 (1 µM) for 1 h and then treated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured using a migration assay (n = 4). (B-C) H1299 and A549 cells were treated with CXCL14 (30 ng/ml) and the phosphorylation of PLCβ3, PKCα, and c-Src was examined using Western blot (n = 4). (D) H1299 and A549 cells were pretreated with U73122 or GF109203X for 1 h and then treated with CXCL14 (30 ng/ml) for 15 min. Phosphorylation of c-Src was measured using Western blot (n = 4). (E, F) H1299 cells were pretreated with U73122, GF109203X or PP2 for 1 h and then incubated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured with migration and wound healing assays (n = 4). (G, H) H1299 and A549 cells were pretreated with U73122, GF109203X or PP2 for 1 h and then treated with CXCL14 (30 ng/ml) for 24 h. The expression of EMT markers was examined using Western blot (n = 4). (I) H1299 and A549 cells were transfected with control siRNA or siRNA specific for PLCβ3, PKCα, or c-Src, and transfection efficiency was measured by Western blot (n = 4). (J, K) H1299 cells were transfected with control siRNA or siRNA specific for PLCβ3, PKCα, or c-Src and then incubated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured using migration and wound healing assays (n = 4). (L) H1299 and A549 cells were transfected with control siRNA or siRNA specific for ACKR2 siRNA and incubated with CXCL14 (30 ng/ml) for 15 min. The phosphorylation of PLCβ3, PKCα, and c-Src was examined using Western blot (n = 4). Untreated cells and cells transfected with control siRNA were used as controls.

Article Snippet: Lentivirus transduction and generation of stable cell lines CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Migration, Marker, Expressing, Phospho-proteomics, Western Blot, Incubation, Transfection, Control

Journal: International journal of biological sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway.

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: Figure 5. CXCL14 promotes cell migration and EMT marker expression through the IKKα, IκBα, and p65 in lung cancer cells. (A, B) H1299 and A549 cells were treated with CXCL14 (30 ng/ml) to detect the phosphorylation of IKKα, IκBα, and p65 by using Western blot (n = 4). (C) H1299 and A549 cells were pretreated with PP2 for 1 h and treated with CXCL14 (30 ng/ml) for 15 min. The phosphorylation of IKKα was measured using Western blot (n = 4). (D, E) H1299 cells were pretreated with TPCK (1 µM) or BAY11-7082 (0.6 µM) for 1 h and incubated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured through migration and wound healing assays (n = 4). (F, G) H1299 and A549 cells were pretreated with TPCK or BAY11-7082 for 1 h and incubated with CXCL14 (0 or 30 ng/ml) for 24 h. The expression of EMT markers was examined by Western blot (n = 4). (H) H1299 and A549 cells were transfected with control siRNA or siRNA specific for p65 siRNA, and transfection efficiency was measured using Western blot (n = 4). (I, J) H1299 cells were transfected with siRNA specific for p65 and treated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured through migration and wound healing assays (n = 4). Untreated cells and cells transfected with control siRNA were used as controls.

Article Snippet: Lentivirus transduction and generation of stable cell lines CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Migration, Marker, Expressing, Phospho-proteomics, Western Blot, Incubation, Transfection, Control

Journal: International journal of biological sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway.

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: Figure 6. CXCL14 promotes NF-κB transcriptional activation via PLCβ3, PKCα, c-Src, IKKα, and IκBα in lung cancer cells. (A, B) H1299 and A549 cells were pretreated with U73122, GF109203X, or PP2 for 1 h and incubated with CXCL14 (30 ng/ml) for 1 h. The phosphorylation of p65 was examined using Western blot (n = 4). (C, D) H1299 and A549 cells were pretreated with TPCK or BAY11-7082 for 1 h and incubated with CXCL14 (30 ng/ml) for 1 h. The phosphorylation of p65 was examined using Western blot (n = 4). (E) H1299 and A549 cells were pretreated with U73122, GF109203X, PP2, TPCK or BAY11-7082 for 1 h and incubated with CXCL14 (30 ng/ml) for 3 h. Cells were stained with anti-p65 antibody and analyzed under an immunofluorescence microscope. The nuclei were counterstained with DAPI. Representative microscopic images are shown. Scale bar = 20 µm. (F) H1299 and A549 cells were transfected with an NF-κB promoter reporter plasmid for 24 h, treated with CXCL14 (1–30 ng/ml) for another 24 h, and evaluated in terms of luciferase activity (n = 4). (G) H1299 and A549 cells were transfected with an NF-κB promoter reporter plasmid for 24 h and pretreated with U73122, GF109203X, PP2, TPCK or BAY11-7082 for 1 h. Cells were incubated with CXCL14 (30 ng/ml) for another 24 h and assessed in terms of luciferase activity (n = 4). Untreated cells were used as controls.

Article Snippet: Lentivirus transduction and generation of stable cell lines CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Activation Assay, Incubation, Phospho-proteomics, Western Blot, Staining, Immunofluorescence, Microscopy, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: International journal of biological sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway.

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: Figure 7. CXCL14 promotes tumor metastasis in the orthotopic model. (A, B) The transfection effects of CXCL14 (OV or KD) in H1299 and A549 cells were examined by Western blot (n = 4). (C, D) The effects of transfection in H1299 and A549 cells were measured using a quantitative real-time polymerase chain reaction (n = 4). (E, F) Cell proliferation of CXCL14-OV and CXCL14-KD cells was examined using a CCK-8 assay (n = 4). (G, H) Cell migration of CXCL14-OV and CXCL14-KD H1299 cells was measured with migration and wound healing assays (n = 4). (I) Schematic illustration of the orthotopic model. (J) Representative images of lung metastases of H1299, CXCL14-OV and CXCL14-KD H1299 vector cells were measured with an in vivo imaging system. The bottom panel showed ex vivo lung tumor images and luminescence intensity of each tumor (n = 4). (K) Representative H&E staining of metastatic lung tumor nodules in each group (n = 4). Scale bar = 4 mm. (L) Representative IHC and H&E staining of orthotopic lung tumor in each group (n = 4). Scale bar = 100 µm.

Article Snippet: Lentivirus transduction and generation of stable cell lines CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Transfection, Western Blot, Real-time Polymerase Chain Reaction, CCK-8 Assay, Migration, Plasmid Preparation, In Vivo Imaging, Ex Vivo, Staining

Journal: International journal of biological sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway.

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: Figure 8. Schematic mechanism of CXCL14 promotes tumor metastasis in lung cancer. The ligand CXCL14 stimulates the atypical chemokine receptor 2 (ACKR2) to activate PLCβ3, PKCα, and c-Src. The phosphorylation of phospholipase Cβ3 (PLCβ3), protein kinase Cα (PKCα), and proto-oncogene c-Src (c-Src) signaling pathways activates IκB kinase α (IKKα) and NF-κB inhibitor α (IκBα) to release nuclear factor-κB (NF-κB). The nuclear translocation of NF-κB promotes the expression of EMT protein and cell migration that leads to tumor metastasis.

Article Snippet: Lentivirus transduction and generation of stable cell lines CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Phospho-proteomics, Protein-Protein interactions, Translocation Assay, Expressing, Migration

Journal: International Journal of Biological Sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: CXCL14 is overexpressed in lung cancer. (A) Difference in CXCL14 expression between normal and lung adenocarcinoma (LUAD) tissues was analyzed by meta-analysis using data downloaded from Lung Cancer Explorer (LCE). (B-D) The gene expression of CXCL14 in normal lung and LUAD obtained from The Cancer Genome Atlas (TCGA). (E, F) CXCL14 mRNA expression in NSCLC and normal lung tissues in GSE18842 and GSE11969 from the Gene Expression Omnibus (GEO) database. (G) Expression of CXCL14 in normal and tumor lung tissues stained with immunohistochemistry (IHC) and different stages was analyzed using the H score (n = 8). Scale bar = 200 μm. (H) The expression of the CXCL14 protein in MRC-5, H1299, and A549 cells was examined using Western blot (n = 4). (I) CXCL14 secretion was examined in MRC-5, H1299, and A549 cells using an enzyme-linked immunosorbent assay (ELISA; n = 4). Normal tissues and MRC-5 cells were used as controls.

Article Snippet: CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Expressing, Gene Expression, Staining, Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Biological Sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: CXCL14 promotes migration and regulates the epithelial mesenchymal transition in lung cancer cells. (A, B) H1299 cells were treated with CXCL14 (1-30 ng/ml) for 24 h, cell migration was measured with migration and wound healing assays (n = 4). (C) H1299 and A549 cells were treated with CXCL14 (1-30 ng/ml) for 24 h, cell viability was evaluated using a CCK-8 assay (n = 4). (D) Expression of CDH2 mRNA in NSCLC and normal lung tissue in GSE18842 from the GEO database. (E) Correlation analysis of CXCL14 and CDH2 in GSE18842. (F) After treatment with CXCL14 (30 ng/ml) for 24 h, the expression levels of E-cadherin, ZO-1, N-cadherin and vimentin were determined in H1299 and A549 cells on immunofluorescence (IF)-stained slides. Scale bar = 20 μm. (G, H) After incubation with CXCL14 (1-30 ng/ml) for 24 h, the expression of epithelial mesenchymal transition (EMT) markers in the H1299 and A549 cells were examined by Western blot (n = 4). Untreated cells were used as controls.

Article Snippet: CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Migration, CCK-8 Assay, Expressing, Immunofluorescence, Staining, Incubation, Western Blot

Journal: International Journal of Biological Sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: ACKR2 is overexpressed and regulates migration in lung cancer. (A-C) Differences in ACKR2 , CXCR4 , and GPR85 expression between normal and lung adenocarcinoma (LUAD) tissues were determined through meta-analysis. (D) The expression levels of ACKR2 in immunohistochemistry (IHC)-stained normal and different stage of tumor lung tissues were analyzed using the H score (n = 8). Scale bar = 200 μm. (E) The expression of the ACKR2 protein in MRC-5, H1299 and A549 cells was examined by Western blot. Data are shown as fold of MRC-5 (n = 4). (F, H) H1299 cells were transfected with control siRNA or specific ACKR2, CXCR4, and GPR85 siRNA for 24 h and incubated with CXCL14 (30 ng/ml) for another 24 h (n = 4). Cell migration was assessed through migration and wound healing assays. (G, I) H1299 cells were incubated with IgG or ACKR2, CXCR4, and GPR85 neutralized antibodies (1 μg/ml) for 1 h and incubated with CXCL14 (30 ng/ml) for another 24 h (n = 4). Cell migration was assessed through migration and wound healing assays. Normal tissues, MRC-5 cells, cells transfected with control siRNA, and incubated with IgG were used as controls.

Article Snippet: CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Migration, Expressing, Immunohistochemistry, Staining, Western Blot, Transfection, Control, Incubation

Journal: International Journal of Biological Sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: CXCL14 promotes cell migration and EMT marker expression via ACKR2 and PLCβ3, PKCα and c-Src in lung cancer cells. (A) A549 cells were pretreated with U73122 (3 μM), GF109203X (1 μM) or PP2 (1 μM) for 1 h and then treated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured using a migration assay (n = 4). (B-C) H1299 and A549 cells were treated with CXCL14 (30 ng/ml) and the phosphorylation of PLCβ3, PKCα, and c-Src was examined using Western blot (n = 4). (D) H1299 and A549 cells were pretreated with U73122 or GF109203X for 1 h and then treated with CXCL14 (30 ng/ml) for 15 min. Phosphorylation of c-Src was measured using Western blot (n = 4). (E, F) H1299 cells were pretreated with U73122, GF109203X or PP2 for 1 h and then incubated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured with migration and wound healing assays (n = 4). (G, H) H1299 and A549 cells were pretreated with U73122, GF109203X or PP2 for 1 h and then treated with CXCL14 (30 ng/ml) for 24 h. The expression of EMT markers was examined using Western blot (n = 4). (I) H1299 and A549 cells were transfected with control siRNA or siRNA specific for PLCβ3, PKCα, or c-Src, and transfection efficiency was measured by Western blot (n = 4). (J, K) H1299 cells were transfected with control siRNA or siRNA specific for PLCβ3, PKCα, or c-Src and then incubated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured using migration and wound healing assays (n = 4). (L) H1299 and A549 cells were transfected with control siRNA or siRNA specific for ACKR2 siRNA and incubated with CXCL14 (30 ng/ml) for 15 min. The phosphorylation of PLCβ3, PKCα, and c-Src was examined using Western blot (n = 4). Untreated cells and cells transfected with control siRNA were used as controls.

Article Snippet: CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Migration, Marker, Expressing, Phospho-proteomics, Western Blot, Incubation, Transfection, Control

Journal: International Journal of Biological Sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: CXCL14 promotes cell migration and EMT marker expression through the IKKα, IκBα, and p65 in lung cancer cells. (A, B) H1299 and A549 cells were treated with CXCL14 (30 ng/ml) to detect the phosphorylation of IKKα, IκBα, and p65 by using Western blot (n = 4). (C) H1299 and A549 cells were pretreated with PP2 for 1 h and treated with CXCL14 (30 ng/ml) for 15 min. The phosphorylation of IKKα was measured using Western blot (n = 4). (D, E) H1299 cells were pretreated with TPCK (1 μM) or BAY11-7082 (0.6 μM) for 1 h and incubated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured through migration and wound healing assays (n = 4). (F, G) H1299 and A549 cells were pretreated with TPCK or BAY11-7082 for 1 h and incubated with CXCL14 (0 or 30 ng/ml) for 24 h. The expression of EMT markers was examined by Western blot (n = 4). (H) H1299 and A549 cells were transfected with control siRNA or siRNA specific for p65 siRNA, and transfection efficiency was measured using Western blot (n = 4). (I, J) H1299 cells were transfected with siRNA specific for p65 and treated with CXCL14 (30 ng/ml) for 24 h. Cell migration was measured through migration and wound healing assays (n = 4). Untreated cells and cells transfected with control siRNA were used as controls.

Article Snippet: CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Migration, Marker, Expressing, Phospho-proteomics, Western Blot, Incubation, Transfection, Control

Journal: International Journal of Biological Sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: CXCL14 promotes NF-κB transcriptional activation via PLCβ3, PKCα, c-Src, IKKα, and IκBα in lung cancer cells. (A, B) H1299 and A549 cells were pretreated with U73122, GF109203X, or PP2 for 1 h and incubated with CXCL14 (30 ng/ml) for 1 h. The phosphorylation of p65 was examined using Western blot (n = 4). (C, D) H1299 and A549 cells were pretreated with TPCK or BAY11-7082 for 1 h and incubated with CXCL14 (30 ng/ml) for 1 h. The phosphorylation of p65 was examined using Western blot (n = 4). (E) H1299 and A549 cells were pretreated with U73122, GF109203X, PP2, TPCK or BAY11-7082 for 1 h and incubated with CXCL14 (30 ng/ml) for 3 h. Cells were stained with anti-p65 antibody and analyzed under an immunofluorescence microscope. The nuclei were counterstained with DAPI. Representative microscopic images are shown. Scale bar = 20 μm. (F) H1299 and A549 cells were transfected with an NF-κB promoter reporter plasmid for 24 h, treated with CXCL14 (1-30 ng/ml) for another 24 h, and evaluated in terms of luciferase activity (n = 4). (G) H1299 and A549 cells were transfected with an NF-κB promoter reporter plasmid for 24 h and pretreated with U73122, GF109203X, PP2, TPCK or BAY11-7082 for 1 h. Cells were incubated with CXCL14 (30 ng/ml) for another 24 h and assessed in terms of luciferase activity (n = 4). Untreated cells were used as controls.

Article Snippet: CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Activation Assay, Incubation, Phospho-proteomics, Western Blot, Staining, Immunofluorescence, Microscopy, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: International Journal of Biological Sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: CXCL14 promotes tumor metastasis in the orthotopic model. (A, B) The transfection effects of CXCL14 (OV or KD) in H1299 and A549 cells were examined by Western blot (n = 4). (C, D) The effects of transfection in H1299 and A549 cells were measured using a quantitative real-time polymerase chain reaction (n = 4). (E, F) Cell proliferation of CXCL14-OV and CXCL14-KD cells was examined using a CCK-8 assay (n = 4). (G, H) Cell migration of CXCL14-OV and CXCL14-KD H1299 cells was measured with migration and wound healing assays (n = 4). (I) Schematic illustration of the orthotopic model. (J) Representative images of lung metastases of H1299, CXCL14-OV and CXCL14-KD H1299 vector cells were measured with an in vivo imaging system. The bottom panel showed ex vivo lung tumor images and luminescence intensity of each tumor (n = 4). (K) Representative H&E staining of metastatic lung tumor nodules in each group (n = 4). Scale bar = 4 mm. (L) Representative IHC and H&E staining of orthotopic lung tumor in each group (n = 4). Scale bar = 100 μm.

Article Snippet: CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Transfection, Western Blot, Real-time Polymerase Chain Reaction, CCK-8 Assay, Migration, Plasmid Preparation, In Vivo Imaging, Ex Vivo, Staining

Journal: International Journal of Biological Sciences

Article Title: CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway

doi: 10.7150/ijbs.79438

Figure Lengend Snippet: Schematic mechanism of CXCL14 promotes tumor metastasis in lung cancer. The ligand CXCL14 stimulates the atypical chemokine receptor 2 (ACKR2) to activate PLCβ3, PKCα, and c-Src. The phosphorylation of phospholipase Cβ3 (PLCβ3), protein kinase Cα (PKCα), and proto-oncogene c-Src (c-Src) signaling pathways activates IκB kinase α (IKKα) and NF-κB inhibitor α (IκBα) to release nuclear factor-κB (NF-κB). The nuclear translocation of NF-κB promotes the expression of EMT protein and cell migration that leads to tumor metastasis.

Article Snippet: CXCL14 human-tagged ORF clone lentiviral particle was purchased from ORiGENE (Rockville, MD, USA).

Techniques: Phospho-proteomics, Protein-Protein interactions, Translocation Assay, Expressing, Migration

Journal: Oncotarget

Article Title: A novel multi-target RNAi adenovirus inhibits hepatoma cell proliferation, migration, and induction of angiogenesis

doi: 10.18632/oncotarget.9531

Figure Lengend Snippet: a. VEGFR2, CCR1, and EpCAM expression of HCC tissues and adjacent non-tumor liver tissue. Localization of VEGF, CCR1, and EpCAM in tissues were determined immunochemically using specific antibodies as described in Materials and Methods. Scale bar, 50 μm; original magnifications, ×400. N, non-tumor liver tissue; T, tumor. b. Western blot analysis of VEGFR2, CCR1, and EpCAM expression of hepatoma cell lines. c. Flow cytometric analysis of cell-surface VEGFR2, CCR1, and EpCAM expression on Huh7 cells. Open heavy-lined and filled histograms represent those with the test antibody and the control IgG, respectively. The representative results from three independent experiments are shown.

Article Snippet: Membranes were subsequently blocked with 5% non-fat milk and incubated overnight at 4°C with polyclonal rabbit anti-human CCR1 antibody (Boster), polyclonal rabbit anti-human VEGFR2 antibody (Boster), or mouse anti-human EpCAM antibody (Boster).

Techniques: Expressing, Western Blot, Control

Journal: Oncotarget

Article Title: A novel multi-target RNAi adenovirus inhibits hepatoma cell proliferation, migration, and induction of angiogenesis

doi: 10.18632/oncotarget.9531

Figure Lengend Snippet: a. shRNA transcripts of VEGFR2, CCR1, and EpCAM were amplified from corresponding single shRNA-expressing vectors, including the H1.1 promoter, shRNA-expressing nucleotides, and terminator. More transcript sequences, including the Bgl II restriction site (5,775 bp) to H1.1 promoter site (6,064 bp) and terminator (1 bp) to NdeI restriction site (261 bp), were amplified, respectively, with VEGFR2 and EpCAM shRNA transcript from corresponding single shRNA-expressing vectors for ligation with Bgl II and NdeI double-digested pRNAT-H1.1 vector. b. shRNA transcripts of VEGFR2, CCR1, and EpCAM were sequentially ligated, and a large shRNA fragment shVCE (1.4 kb) was obtained. c. pRNAT-shVCE was generated by combination of shVCE and pRNAT-H1.1/shuttle via BglII and NdeI restriction enzyme sites. d. Clone 3 of pRNAT-shVCE was selected for further study and inserted into a pRNAT-H1.1/shuttle. e. A multiple shRNA expressing vector targeting VEGFR2, CCR1, and EpCAM was constructed as shown in the diagram.

Article Snippet: Membranes were subsequently blocked with 5% non-fat milk and incubated overnight at 4°C with polyclonal rabbit anti-human CCR1 antibody (Boster), polyclonal rabbit anti-human VEGFR2 antibody (Boster), or mouse anti-human EpCAM antibody (Boster).

Techniques: shRNA, Amplification, Expressing, Ligation, Plasmid Preparation, Generated, Construct

Journal: Oncotarget

Article Title: A novel multi-target RNAi adenovirus inhibits hepatoma cell proliferation, migration, and induction of angiogenesis

doi: 10.18632/oncotarget.9531

Figure Lengend Snippet: a. The mRNA expression levels of VEGFR2, CCR1, and EpCAM were analyzed by quantitative PCR at different times after vector transfection. b. Protein expression levels were analyzed by western blot at different times after vector transfection. Statistical analysis for the western blot data is shown on the right. Data are expressed as mean ± SD. * denotes >60% knockdown efficiency versus non-treated Huh7 cells.

Article Snippet: Membranes were subsequently blocked with 5% non-fat milk and incubated overnight at 4°C with polyclonal rabbit anti-human CCR1 antibody (Boster), polyclonal rabbit anti-human VEGFR2 antibody (Boster), or mouse anti-human EpCAM antibody (Boster).

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