Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae–induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045r

Figure Lengend Snippet: Figure 3. PI3K mediates TGF-b1–dependent activation of TRPV4, whereas p38 and Rho are activated downstream of TRPV4. A–C) NHLF cells were preincubated with indicated concentrations of PI3K inhibitor (LY294002; A), p38 MAPK inhibitor (SB203580; B), and Rho kinase inhibitor (Y27632; C) for 30 min, followed by treatment with TGF-b1 (2 ng/ml; 48 h), and a-SMA was determined by Western blotting. Note that all the 3 inhibitors blocked TGF-b1–induced a-SMA induction. D, E) PI3K P110a protein (D) and p38 MAPK (E) was knocked down in NHLFs by transfecting them with corresponding siRNA (100 nM). NHLFs transfected with nonspecific (NS) siRNA were used as control. Twenty-four hours after transfection, NHLFs were treated with TGF-b1 (2 ng/ml; 48 h) and a-SMA expression was analyzed by Western blotting. F, G, J) Effect of TRPV4 antagonist on the activation of AKT, p38 MAPK, and Rho. NHLFs were preincubated with RN1734 (30 mM) and treated with TGF-b1 for indicated time points. Western blot analysis showing the phosphorylation of AKT (F) and phosphorylation of p38 MAPK (30 min; G) and Rho activation (J ). Rho activity was measured by RhoA activation assay kit (Cytoskeleton) 60 min after TGF-b1 stimulation. Total Rho in the lysates was determined by Western blotting. Blots were stripped and reprobed with GAPDH Ab and quantified by using AlphaView software. H, I), Fluoroscence images showing the effect of PI3K inhibitor on TGF- b1–mediated TRPV4 membrane translocation. NHLFs were transfected with GFP-tagged TRPV4 construct and translocation of TRPV4 to the membrane was analyzed in response to TGF-b1 treatment (2 ng/ml) with or without LY (50 mM) over time (0, 0.5, and 2 h). Number of cells with TRPV4-GFP at the plasma membrane was counted without knowledge of the treatment groups and expressed as percentage. Results are means 6 SEM from at least 3 independent experiments (A–C, I, J). *P , 0.05; **P , 0.01.

Article Snippet: The following chemicals and reagents were purchased commercially: RN1734 (selective TRPV4 inhibitor) (20, 23), SB203580 (p38 inhibitor, binds to the ATP-binding pocket) (24, 25), and Y27632 dihydrochloride (Rho kinase inhibitor; all from Tocris Bioscience, Minneapolis, MN, USA) (26, 27); LY294002 (PI3K inhibitor; Cayman Chemicals, Ann Arbor, MI, USA) (28, 29); TGF-b1 (R&D Systems, Minneapolis, MN, USA); PAI-1, phospho-Akt, phospho-p38 MAPK, p38 MAPK, PI3K, lamin, and a-tubulin Abs (Cell Signaling Technology, Danvers, MA, USA); a-SMA antibody and GSK1016790A (Sigma-Aldrich, St. Louis, MO, USA); FN Ab (Abcam, Cambridge, MA, USA); MRTF-A Ab (Santa Cruz Biotechnology Santa Cruz, CA, USA); glyceraldehyde-3-phosphatedehydrogenase (GAPDH;Fitzgerald, Acton, MA, USA); all secondary Abs (Jackson ImmunoResearch, WestGrove, PA,USA); Fluo-4 (ThermoFisher Scientific,Waltham, MA, USA); nontargeting small interfering RNA (siRNA) and specific siRNA for TRPV4 (Dharmacon, Lafayette, CO, USA); specific siRNA for PI3K and p38 (Cell Signaling Technology); siLentFect lipid reagent (Bio-Rad, Hercules, CA, USA); RhoA G-LISA activation kit (Cytoskeleton, Denver, CO, USA), Diff Quik Stain Set (Siemens, Newark, DE, USA); subcellular protein fractionation kit for cultured cells (Thermo Fisher Scientific); and transcriptor first strand cDNA synthesis kit and light cycler 480 SYBR Green I Master Mix (Roche, Indianapolis, IN, USA).

Techniques: Activation Assay, Western Blot, Transfection, Control, Expressing, Phospho-proteomics, Activity Assay, Software, Membrane, Translocation Assay, Construct, Clinical Proteomics

Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae–induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045r

Figure Lengend Snippet: Figure 5. TRPV4 regulates TGF-b1–mediated fibrotic gene expression. A–C) NHLFs were treated with TGF-b1 (2 ng/ml; 48 h) with or without RN1734 (30 mM) and the transcripts of SM22 (A, B) and MRTF-A (C) were analyzed by real-time PCR. D, F) MRTF-A protein was determined by Western blotting (D) and TRPV4-dependent MRTF-A nuclear translocation (F) to the membrane in response to TGF-b1 (2 ng/ml; 24 h) in the presence or absence of TRPV4 inhibitor RN1734 (30 mM) was analyzed by Western blotting in nuclear fractions. Blot was stripped and reprobed with lamin Ab to confirm equal loading. B, E) DHLFs exhibit higher basal and TGF-b1–mediated SM22 mRNA and MRTF-A protein expression. NHLFs and DHLFs were treated with TGF-b1 (2 ng/ml; 48 h) and (B) SM22 transcript was analyzed by real-time PCR (E) MRTF-A protein was determined by Western blotting. G) TGF-b1–mediated MRTF-A expression is sensitive to Rho kinase inhibition. NHLFs were treated with TGF-b1 (2 ng/ml; 48 h) with or without Y27632 (10 mM) and MRTF-A protein was determined by Western blotting. Blots were stripped and reprobed with GAPDH Ab and were quantified by using AlphaView software. Graphs represent means 6 SEM from at least 3 independent experiments. *P , 0.05; **P , 0.01; ***P , 0.001.

Article Snippet: The following chemicals and reagents were purchased commercially: RN1734 (selective TRPV4 inhibitor) (20, 23), SB203580 (p38 inhibitor, binds to the ATP-binding pocket) (24, 25), and Y27632 dihydrochloride (Rho kinase inhibitor; all from Tocris Bioscience, Minneapolis, MN, USA) (26, 27); LY294002 (PI3K inhibitor; Cayman Chemicals, Ann Arbor, MI, USA) (28, 29); TGF-b1 (R&D Systems, Minneapolis, MN, USA); PAI-1, phospho-Akt, phospho-p38 MAPK, p38 MAPK, PI3K, lamin, and a-tubulin Abs (Cell Signaling Technology, Danvers, MA, USA); a-SMA antibody and GSK1016790A (Sigma-Aldrich, St. Louis, MO, USA); FN Ab (Abcam, Cambridge, MA, USA); MRTF-A Ab (Santa Cruz Biotechnology Santa Cruz, CA, USA); glyceraldehyde-3-phosphatedehydrogenase (GAPDH;Fitzgerald, Acton, MA, USA); all secondary Abs (Jackson ImmunoResearch, WestGrove, PA,USA); Fluo-4 (ThermoFisher Scientific,Waltham, MA, USA); nontargeting small interfering RNA (siRNA) and specific siRNA for TRPV4 (Dharmacon, Lafayette, CO, USA); specific siRNA for PI3K and p38 (Cell Signaling Technology); siLentFect lipid reagent (Bio-Rad, Hercules, CA, USA); RhoA G-LISA activation kit (Cytoskeleton, Denver, CO, USA), Diff Quik Stain Set (Siemens, Newark, DE, USA); subcellular protein fractionation kit for cultured cells (Thermo Fisher Scientific); and transcriptor first strand cDNA synthesis kit and light cycler 480 SYBR Green I Master Mix (Roche, Indianapolis, IN, USA).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Translocation Assay, Membrane, Expressing, Inhibition, Software

Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae–induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045r

Figure Lengend Snippet: Figure 7. TRPV4-mediated signal transduction downstream of TGF-b1. NHLF cells were treated with TGF-b1 (2 ng/ml) for 48 h or indicated times in the presence or absence of SB203580 (10 mM), Y27632 (10 mM; A), and a-SMA, SM22, and PAI-1 transcripts were analyzed by real-time PCR (B, C, E) PAI-1 protein; (B, C, F) FN protein was determined by Western blotting. F) p38 MAPK is downstream of Rho kinase. NHLF cells were treated with TGF-b1 (2 ng/ml) for 30 min in the presence or absence of Y27632 (10 mM), and phosphorylation of p38 was determined and quantified. Blots were stripped and reprobed with GAPDH Ab and were quantified by using AlphaView software. Graphs represent means 6 SEM from at least 3 independent experiments (A, D, E, F). *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: The following chemicals and reagents were purchased commercially: RN1734 (selective TRPV4 inhibitor) (20, 23), SB203580 (p38 inhibitor, binds to the ATP-binding pocket) (24, 25), and Y27632 dihydrochloride (Rho kinase inhibitor; all from Tocris Bioscience, Minneapolis, MN, USA) (26, 27); LY294002 (PI3K inhibitor; Cayman Chemicals, Ann Arbor, MI, USA) (28, 29); TGF-b1 (R&D Systems, Minneapolis, MN, USA); PAI-1, phospho-Akt, phospho-p38 MAPK, p38 MAPK, PI3K, lamin, and a-tubulin Abs (Cell Signaling Technology, Danvers, MA, USA); a-SMA antibody and GSK1016790A (Sigma-Aldrich, St. Louis, MO, USA); FN Ab (Abcam, Cambridge, MA, USA); MRTF-A Ab (Santa Cruz Biotechnology Santa Cruz, CA, USA); glyceraldehyde-3-phosphatedehydrogenase (GAPDH;Fitzgerald, Acton, MA, USA); all secondary Abs (Jackson ImmunoResearch, WestGrove, PA,USA); Fluo-4 (ThermoFisher Scientific,Waltham, MA, USA); nontargeting small interfering RNA (siRNA) and specific siRNA for TRPV4 (Dharmacon, Lafayette, CO, USA); specific siRNA for PI3K and p38 (Cell Signaling Technology); siLentFect lipid reagent (Bio-Rad, Hercules, CA, USA); RhoA G-LISA activation kit (Cytoskeleton, Denver, CO, USA), Diff Quik Stain Set (Siemens, Newark, DE, USA); subcellular protein fractionation kit for cultured cells (Thermo Fisher Scientific); and transcriptor first strand cDNA synthesis kit and light cycler 480 SYBR Green I Master Mix (Roche, Indianapolis, IN, USA).

Techniques: Transduction, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Software

Journal: Oncology Reports

Article Title: Decorin suppresses lung metastases of murine osteosarcoma

doi: 10.3892/or.19.6.1533

Figure Lengend Snippet: Figure 1. The expression of decorin in Dunn and LM8 murine osteosarcoma cell lines. (A) The decorin gene expression detected by RT-PCR. (B) Decorin protein expression detected by Western blot analysis. These data confirmed that the expression level of decorin in the the LM8 cells was lower than that of the Dunn cells.

Article Snippet: Recombinant human decorin protein (R&D systems, AF143-DE) was used as a positive control.

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Oncology Reports

Article Title: Decorin suppresses lung metastases of murine osteosarcoma

doi: 10.3892/or.19.6.1533

Figure Lengend Snippet: Figure 2. The isolation of decorin expressing LM8 cell lines. After isolation of the 2 types of subclones of LM8, LM8-DCN, which stably expresses the decorin protein and LM8-mock, the decorin-protein expression, was examined using Western blot analysis. There was no difference in the expression of the mouse decorin observed between LM8-mock and LM8-DCN (A). However, the human decorin expression was found only in LM8-DCN (B).

Article Snippet: Recombinant human decorin protein (R&D systems, AF143-DE) was used as a positive control.

Techniques: Isolation, Expressing, Stable Transfection, Western Blot

Journal: Oncology Reports

Article Title: Decorin suppresses lung metastases of murine osteosarcoma

doi: 10.3892/or.19.6.1533

Figure Lengend Snippet: Figure 4. In vivo effect of decorin on the pulmonary metastasis of murine osteosarcoma. (A) A microphotograph of pulmonary metastases in mice with LM8-DCN and LM8-mock. (B) The inhibitory effect of decorin on lung metastasis in murine osteosarcoma. The mean number of lung metastatic nodules 4 weeks after tumor cell inoculation was significantly reduced in mice inoculated with LM8-DCN cells compared to those inoculated with LM8 or LM8-mock cells. The mean number of nodules on the lung at their maximum dimensions was 27.4±20.8 with LM8, 29.8±22.7 with LM8-mock and 2.4±3.03 with LM8-DCN. *P<0.001 as compared to the LM8 and LM8- mock.

Article Snippet: Recombinant human decorin protein (R&D systems, AF143-DE) was used as a positive control.

Techniques: In Vivo

Journal: Oncology Reports

Article Title: Decorin suppresses lung metastases of murine osteosarcoma

doi: 10.3892/or.19.6.1533

Figure Lengend Snippet: Figure 7. The inhibitory effect of decorin on cell motility and invasion in vitro. (A) The inhibitory effect of decorin on cell motility. After the suspension of each cell line followed by a 12 h incubation at 37˚C in 5% CO2, the migrant cells on the undersurface were fixed and stained with diff-Quick stain (Baxter). The attached cells on the undersurface of the membranes were counted from 10 random microscopic fields (magnification, x100). (B) The inhibitory effect of decorin on cell invasion. After the suspension of each cell line followed by a 24 h incubation at 37˚C in 5% CO2, the migrant cells on the undersurface were fixed and stained with diff-Quick stain. The adherent cells on the membranes were counted from five random microscopic fields (magnification, x100). Decorin inhibits the LM8 cell motility and invasion ability (*P<0.05 using the Mann-Whitney test).

Article Snippet: Recombinant human decorin protein (R&D systems, AF143-DE) was used as a positive control.

Techniques: In Vitro, Suspension, Incubation, Staining, Diff-Quik, MANN-WHITNEY

Journal: Cancer Science

Article Title: Prostate cancer antigen‐1 contributes to cell survival and invasion though discoidin receptor 1 in human prostate cancer

doi: 10.1111/j.1349-7006.2007.00655.x

Figure Lengend Snippet: Prostate cancer antigen (PCA)‐1 knockdown‐induced apoptosis in PC3 cells. (a) PC3 cells were transfected with or without control RNA or PCA‐1 small interfering RNA (siRNA). After 72 h cultivation, the mRNA and protein expression of PCA‐1, Bcl‐xl (whole‐cell lysates), and cytochrome c (cytoplasmic fraction) were examined by reverse transcription–polymerase chain reaction (RT‐PCR) and western blotting. (b) After transfection with control RNA or PCA‐1 siRNA, cells were fixed and incubated with fluorescein isothiocyanate‐conjugated terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) reaction mixture. Images of nuclear fluorescence typical of apoptotic features were collected by fluorescence microscopy and the percentages of TUNEL‐positive apoptotic cells were calculated (upper and lower left panel). After the same treatment, cells were harvested, and the levels of full‐length and cleaved poly ADP‐ribose polymerase (PARP) were analyzed by western blotting using anti‐PARP antibody (lower right panel). Glyceraldehyde‐3‐phosphate dehydrogenase or actin was used as a housekeeping gene in RT‐PCR or western blotting.

Article Snippet: The cells were seeded at 10 6 cells per well in 6‐cm plates, then transfected with 100 nM control RNA (Santa Cruz), PCA‐1 siRNA (horseradish peroxidase [HP]‐validated siRNA 1027400; Qiagen) or DDR1 siRNA (Santa Cruz) in the presence or absence of the GFP‐carrying vector pEGFP using lipofectamine (Invitrogen, Tokyo, Japan) in accordance with the manufacturer's protocol.

Techniques: Transfection, Small Interfering RNA, Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation, End Labeling, TUNEL Assay, Fluorescence, Microscopy

Journal: Cancer Science

Article Title: Prostate cancer antigen‐1 contributes to cell survival and invasion though discoidin receptor 1 in human prostate cancer

doi: 10.1111/j.1349-7006.2007.00655.x

Figure Lengend Snippet: Discoidin domain receptor (DDR) is downstream of prostate cancer antigen (PCA)‐1 and contributes to prostate cancer invasion. (a) PC3 cells were transfected with control RNA, or PCA‐1 or DDR1 small interfering RNA (siRNA). After 72 h cultivation, the mRNA and protein expression of PCA‐1, DDR1, and Bcl‐xl was examined by reverse transcription–polymerase chain reaction and western blotting. (b) After transfection with control RNA or siRNA as described in (a), the cells were lysed and immunoprecipitated with anti‐DDR1 antibody. Phosphorylation of DDR1 was analyzed by western blotting using antiphosphotyrosine antibody. (c) After transfection with control RNA or DDR1 siRNA as described in (a), cells were fixed and incubated with fluorescein isothiocyanate‐conjugated terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) reaction mixture. Images of nuclear fluorescence typical of apoptotic features were collected by fluorescence microscopy, and the percentages of TUNEL‐positive apoptotic cells were calculated. (d) At 48 h after transfection with or without PCA‐1 or DDR1 siRNA, cells were placed on matrigel. After 36 h incubation, invading cells were fixed and stained with the Diff Quick staining kit, then counted under a light microscope.

Article Snippet: The cells were seeded at 10 6 cells per well in 6‐cm plates, then transfected with 100 nM control RNA (Santa Cruz), PCA‐1 siRNA (horseradish peroxidase [HP]‐validated siRNA 1027400; Qiagen) or DDR1 siRNA (Santa Cruz) in the presence or absence of the GFP‐carrying vector pEGFP using lipofectamine (Invitrogen, Tokyo, Japan) in accordance with the manufacturer's protocol.

Techniques: Transfection, Small Interfering RNA, Expressing, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Incubation, End Labeling, TUNEL Assay, Fluorescence, Microscopy, Staining, Diff-Quik, Light Microscopy

Journal: Cancer Science

Article Title: Prostate cancer antigen‐1 contributes to cell survival and invasion though discoidin receptor 1 in human prostate cancer

doi: 10.1111/j.1349-7006.2007.00655.x

Figure Lengend Snippet: Prostate cancer antigen (PCA)‐1 overexpression enhanced cancer invasion through upregulation of matrix metalloproteinase (MMP) 9. (a) DU145‐derived clones overexpressing the PCA‐1 gene were selected (DU145 PCA‐1 [A] and [B]), and the protein expression of His‐tagged PCA‐1, discoidin domain receptor (DDR), and Bcl‐xl was examined by western blotting using antibodies against His, DDR1, and Bcl‐xl. (b) DU145 PCA‐1 (A) and (B) cells were transfected with or without PCA‐1 or DDR1 siRNA. After 72 h cultivation, the culture medium was concentrated and the gelatinolytic activity of MMP9 was examined. The mRNA expression of MMP9 was also investigated by reverse transcription–polymerase chain reaction. (c) PC3 cells were transfected with green fluorescent protein (GFP)‐encoding plasmids in the presence of control RNA or PCA‐1 siRNA. DU145 cells and DU145 PCA‐1 (A) and (B) cells were similarly transfected with GFP‐encoding plasmids. After 24 h cultivation, these cells were plated on chorioallantoic membrane, and grafts were collected at 3 days. Tissues were fixed and stained with anti‐GFP; thereafter, the number of invading cancer cells positive for GFP was quantified in three or more randomly selected fields.

Article Snippet: The cells were seeded at 10 6 cells per well in 6‐cm plates, then transfected with 100 nM control RNA (Santa Cruz), PCA‐1 siRNA (horseradish peroxidase [HP]‐validated siRNA 1027400; Qiagen) or DDR1 siRNA (Santa Cruz) in the presence or absence of the GFP‐carrying vector pEGFP using lipofectamine (Invitrogen, Tokyo, Japan) in accordance with the manufacturer's protocol.

Techniques: Over Expression, Derivative Assay, Clone Assay, Expressing, Western Blot, Transfection, Activity Assay, Reverse Transcription, Polymerase Chain Reaction, Membrane, Staining

Journal: Cancer Science

Article Title: Prostate cancer antigen‐1 contributes to cell survival and invasion though discoidin receptor 1 in human prostate cancer

doi: 10.1111/j.1349-7006.2007.00655.x

Figure Lengend Snippet: Amplification of prostate cancer antigen (PCA)‐1‐mediated signals and androgen independence. (a) Androgen‐independent (AI) LNCaP cells were developed under long‐term culture in androgen‐free medium. Protein expression of PCA‐1, discoidin domain receptor (DDR), and Bcl‐xl in LNCaP and AI LNCaP with or without transfection of control RNA or PCA‐1 or DDR1 small interfering RNA (siRNA) (72‐h incubation period) was examined by western blotting. (b) LNCaP and AI LNCaP cells were transfected with control or PCA‐1 or DDR1 siRNA. After 24 h cultivation, cells were plated on chorioallantoic membrane and incubated for 72 h. Tissues were fixed and stained with anti‐green fluorescent protein (GFP); thereafter, the number of invading cancer cells positive for GFP was quantified in three or more randomly selected fields.

Article Snippet: The cells were seeded at 10 6 cells per well in 6‐cm plates, then transfected with 100 nM control RNA (Santa Cruz), PCA‐1 siRNA (horseradish peroxidase [HP]‐validated siRNA 1027400; Qiagen) or DDR1 siRNA (Santa Cruz) in the presence or absence of the GFP‐carrying vector pEGFP using lipofectamine (Invitrogen, Tokyo, Japan) in accordance with the manufacturer's protocol.

Techniques: Amplification, Expressing, Transfection, Small Interfering RNA, Incubation, Western Blot, Membrane, Staining

Journal: Cancer Science

Article Title: Prostate cancer antigen‐1 contributes to cell survival and invasion though discoidin receptor 1 in human prostate cancer

doi: 10.1111/j.1349-7006.2007.00655.x

Figure Lengend Snippet: Expression of prostate cancer antigen (PCA)‐1 and discoidin domain receptor (DDR) in normal prostatic epithelial cells and cancer cells. (a) Immunohistochemical analysis of PCA‐1 and DDR1 in prostatic epithelial and cancer tissues of no (score 0), mild (score 1), moderate (score 2), and high (score 3) intensities. Normal prostatic epithelial tissues were obtained from benign prostatic hypertrophy. NE, normal prostatic epithelial cells; PIN, prostatic intraepithelial neoplasm. (b) To characterize the specificity of antibodies used in the present study, western blotting analysis of PCA‐1 and DDR1 was carried out in a sample of normal prostatic epithelial cells obtained from benign prostatic hypertrophy and four prostate cancer cells. (c) Statistical analysis of the intensities of PCA‐1 and DDR1 in normal prostatic epithelial cells, prostate cancer cells, or precancerous cells (lesions). (d) Kaplan–Meier analysis of hormone‐resistant prostate‐specific antigen recurrence‐free survival in groups of patients with low or moderate (less than score 2) and high (more than score 3) levels of PCA‐1 or DDR1 expression. The follow‐up period was 2000 days.

Article Snippet: The cells were seeded at 10 6 cells per well in 6‐cm plates, then transfected with 100 nM control RNA (Santa Cruz), PCA‐1 siRNA (horseradish peroxidase [HP]‐validated siRNA 1027400; Qiagen) or DDR1 siRNA (Santa Cruz) in the presence or absence of the GFP‐carrying vector pEGFP using lipofectamine (Invitrogen, Tokyo, Japan) in accordance with the manufacturer's protocol.

Techniques: Expressing, Immunohistochemical staining, Western Blot

Journal: Cancer Science

Article Title: Prostate cancer antigen‐1 contributes to cell survival and invasion though discoidin receptor 1 in human prostate cancer

doi: 10.1111/j.1349-7006.2007.00655.x

Figure Lengend Snippet: Schematic presentation of the correlation of prostate cancer antigen (PCA)‐1–discoidin domain receptor (DDR)‐mediated signals with androgen independence (resistance) and cancer progression in human prostate cancer. PCA‐1 and downstream DDR1 and Bclxl or matrix metaloprotease (MMP) 9 signals promoted cancer invasion and survival. PCA‐1‐mediated signals were amplified, associated with hormone resistance and enhance malignant potential of androgen‐independent prostate cancer cells.

Article Snippet: The cells were seeded at 10 6 cells per well in 6‐cm plates, then transfected with 100 nM control RNA (Santa Cruz), PCA‐1 siRNA (horseradish peroxidase [HP]‐validated siRNA 1027400; Qiagen) or DDR1 siRNA (Santa Cruz) in the presence or absence of the GFP‐carrying vector pEGFP using lipofectamine (Invitrogen, Tokyo, Japan) in accordance with the manufacturer's protocol.

Techniques: Amplification

Journal: PLoS ONE

Article Title: Identification of Myeloid Derived Suppressor Cells in Dogs with Naturally Occurring Cancer

doi: 10.1371/journal.pone.0033274

Figure Lengend Snippet: PBMCs from healthy dogs and dogs with cancer were stained for the myeloid marker CD11b, monocytic marker CD14 and MHC II. (A) Representative flow cytometric analysis of forward and side scatter and gated CD11b + CD14 − MHCII − cells from dogs with advanced or metastatic tumors compared to dogs with early stage non-metastatic tumors and healthy control dogs. Plots are representative of dog with advanced metastatic hemangiosarcoma (top), early stage bladder transitional cell carcinoma (middle) and a healthy dog. (B) FACS sorted CD11b + CD14 − MHCII − cells were stained with diff-quick for cell morphology evaluation. A representative example of polymorphonuclear granulocyte morphology of CD11b + CD14 − MHCII − cells is shown at 63× magnification.

Article Snippet: Following indirect staining, cells were washed twice and stained with 0.3 μg of FITC-conjugated rat anti-dog MHCII (clone YKIX334.2, AbD Serotec) and 0.15 μg of the cross-reactive, Alexa fluor 647-conjugated mouse anti-human CD14 antibody (clone TÜK4, AbD Serotec) or isotypes controls at 4°C for 30 min in a dark room according to manufacturer's protocol.

Techniques: Staining, Marker, Control, Diff-Quik

Journal: PLoS ONE

Article Title: Identification of Myeloid Derived Suppressor Cells in Dogs with Naturally Occurring Cancer

doi: 10.1371/journal.pone.0033274

Figure Lengend Snippet: (A) Analysis of average CD11 + CD14 − MHCII − population frequency in dogs with advanced stage or metastatic tumors (n = 30) compared with early stage non-metastatic tumors (n = 15) and control dogs (n = 18). There was a significantly higher percentage of CD11b + CD14 − MHCII − cells in dogs with advanced cancer versus early stage non-metastatic tumors and healthy dogs (36.04% vs. 9.40% and10.24%, respectively. B) Average CD11b + CD14 − MHCII − population frequency in the major cancer subtypes: advanced stage or metastatic sarcomas (n = 18), early stage non-metastatic sarcomas (n = 6), advanced stage or metastatic carcinomas (n = 7) early stage non-metastatic carcinomas (n = 7), advanced stage or metastatic mast cell tumors (n = 5) and early stage non-metastatic mast cell tumors (n = 2) compared with control dogs (n = 18). Significantly elevated percentages were detected in all advanced tumors subtypes relative to early stage tumors and healthy dogs. Percentages of CD11b + CD14 + MHCII − cells were not significant between groups (* indicates P<0.001). Mean ± SEM are shown.

Article Snippet: Following indirect staining, cells were washed twice and stained with 0.3 μg of FITC-conjugated rat anti-dog MHCII (clone YKIX334.2, AbD Serotec) and 0.15 μg of the cross-reactive, Alexa fluor 647-conjugated mouse anti-human CD14 antibody (clone TÜK4, AbD Serotec) or isotypes controls at 4°C for 30 min in a dark room according to manufacturer's protocol.

Techniques: Control

Journal: PLoS ONE

Article Title: Identification of Myeloid Derived Suppressor Cells in Dogs with Naturally Occurring Cancer

doi: 10.1371/journal.pone.0033274

Figure Lengend Snippet: CD11b + CD14 − MHCII − cells were sorted from peripheral blood sample of dogs with cancer and then co-cultured with autologous PBMCs (A, B) or healthy dog PBMCs (C) in the presence of mitogen for 72 hs. Representative examples from a total of eight dogs are shown. The graphs represent proliferative responses after addition of CD11b + CD14 − MHCII − isolated from a single dog with squamous cell carcinoma (3A), prostatic adenocarcinoma (3B) and osteosarcoma (3C). Non-stimulated PBMCs were used as negative control and PBMCs stimulated in absence of CD11b + CD14 − MHCII − cells were used as positive control for proliferation. PBMCs were also co-incubated with PMNs, to control for presence of additional cells (3C, 3D). Proliferative responses were measured by 3 H-thymidine incorporation. CPM, counts per minute. Amount of IFN-γ secretion in the co-culture was determined using canine specific IFN-γ ELISA assay (3D). All experiments were performed in triplicate. Mean ± SEM are shown.

Article Snippet: Following indirect staining, cells were washed twice and stained with 0.3 μg of FITC-conjugated rat anti-dog MHCII (clone YKIX334.2, AbD Serotec) and 0.15 μg of the cross-reactive, Alexa fluor 647-conjugated mouse anti-human CD14 antibody (clone TÜK4, AbD Serotec) or isotypes controls at 4°C for 30 min in a dark room according to manufacturer's protocol.

Techniques: Cell Culture, Isolation, Negative Control, Positive Control, Incubation, Control, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Identification of Myeloid Derived Suppressor Cells in Dogs with Naturally Occurring Cancer

doi: 10.1371/journal.pone.0033274

Figure Lengend Snippet: Facs sorted CD11b + CD14 − MHCII − cells isolated from a dog with osteosarcoma or healthy PBMCs were co-incubated with mitogen-stimulated CD4 + and CD8 + T cells isolated from a healthy dog for 72 hs. No stimulated cells were used as negative control. Proliferative responses were measured by 3 H-thymidine incorporation from experiments performed in triplicate. CPM, counts per minute. Mean ± SEM are shown.

Article Snippet: Following indirect staining, cells were washed twice and stained with 0.3 μg of FITC-conjugated rat anti-dog MHCII (clone YKIX334.2, AbD Serotec) and 0.15 μg of the cross-reactive, Alexa fluor 647-conjugated mouse anti-human CD14 antibody (clone TÜK4, AbD Serotec) or isotypes controls at 4°C for 30 min in a dark room according to manufacturer's protocol.

Techniques: Isolation, Incubation, Negative Control

Journal: PLoS ONE

Article Title: Identification of Myeloid Derived Suppressor Cells in Dogs with Naturally Occurring Cancer

doi: 10.1371/journal.pone.0033274

Figure Lengend Snippet: RT-PCR analysis of FACS purified CD11b + CD14 − MHCII − cells detected expression of ARG1 and iNOS2, as well TGF-β and IL-10 immunosuppressive cytokines. ARG-1 expression was not detected in normal PMNs. CD11b + CD14 − MHCII − cells were isolated from the peripheral blood of a dog with osteosarcoma and PMNs were isolated from a healthy dog. NRT, RNA template in the absence of reverse transcriptase. are representative three experiments.

Article Snippet: Following indirect staining, cells were washed twice and stained with 0.3 μg of FITC-conjugated rat anti-dog MHCII (clone YKIX334.2, AbD Serotec) and 0.15 μg of the cross-reactive, Alexa fluor 647-conjugated mouse anti-human CD14 antibody (clone TÜK4, AbD Serotec) or isotypes controls at 4°C for 30 min in a dark room according to manufacturer's protocol.

Techniques: Reverse Transcription Polymerase Chain Reaction, Purification, Expressing, Isolation, Reverse Transcription