Journal: Frontiers in pharmacology

Article Title: Galectin-1 Facilitates Macrophage Reprogramming and Resolution of Inflammation Through IFN-β.

doi: 10.3389/fphar.2020.00901

Figure Lengend Snippet: FIGURE 3 | Gal-1 shifts the cytokine production of resolution-phase macrophages to promote their reprogramming. (A-G) Peritoneal macrophages were isolated from WT and Gal-1null male mice 48 h PPI and then exposed to vehicle or LPS for 24 h. The concentrations of the following cytokines and chemokines were measured in the culture media by ELISA, n = 4–6: (A) TNF-a, (B) IL-12, (C) IL-6, (D) IL-10, (E) CCL2, and (F) CCL5. (G) TGF-b concentrations in the peritoneal fluids at 48 h were directly measured. (H–K) Peritoneal cells were collected from mice treated with anti-Gal-1 or Iso Ab immunostained as in Figure 2, as well as for TLR4, and analyzed by flow cytometry. Then, TLR4 expression on various leukocyte subsets at 12 and 48 h was determined. (L) Macrophages were isolated from peritoneal exudates of four mice at 48 h PPI and treated with vehicle or Gal-1 (4 mg/ml) for 30 min. Then, macrophages were lysed and their protein content was run by SDS-PAGE and blotted for phospho-STAT1, phospho-STAT3, and actin. Representative images are shown. Full blot images are shown in Figure S8. Altogether, the data represent at least two independent experiments and are presented as mean ± SEM. Statistical significance relative to WT mice was analyzed by two-tailed Student's t-test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The indicated reagents were obtained as follows: ELISA kits for mouse TNF-a, IL-10, IL-6, and IL-12 (Biolegend), and for mouse CCL2 and CCL5 (R&D systems); FITC-conjugated anti-mouse Gr-1, FITC-conjugated anti-mouse Ly6G, PE-conjugated antimouse F4/80, PerCP-conjugated anti-mouse CD11b, PBconjugated anti-mouse Ly6C and PE/Cy7-conjugated antimouse TLR4 (Biolegend); APC-conjugated anti-mouse Tim4 (Miltenyi); Alx647-conjugated anti-mouse IgG, goat antimouse arginase-1, and rabbit anti-IFN-b antibodies (Abcam); rabbit anti-mouse 12/15-lipoxygenase antibody (Cayman Chemical); goat anti-mouse CD11b, goat anti-mouse b-actin and goat anti-PR3 antibodies (Santa Cruz Biotechnology); antip-STAT-1 and anti-p-STAT3 (Cell Signalling); anti-goat and anti-rabbit horseradish peroxidase-conjugated antibodies (Jackson ImmunResearch laboratories).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Cytometry, Expressing, SDS Page, Two Tailed Test

Journal: Frontiers in pharmacology

Article Title: Galectin-1 Facilitates Macrophage Reprogramming and Resolution of Inflammation Through IFN-β.

doi: 10.3389/fphar.2020.00901

Figure Lengend Snippet: FIGURE 7 | IFN-b rescues hampered keys of resolution of inflammation in Gal-1null mice. (A, B) WT and Gal-1null male mice undergoing peritonitis were administered with IFN-b or vehicle 24 h PPI, and their peritoneal exudates were collected 24 h later. Macrophages were isolated from the exudates and exposed to vehicle or LPS for 24 h. (C, D) Peritoneal macrophages were isolated from WT and Gal-1null male mice 66 h PPI. Then, the macrophages were treated with IFN-b or vehicle, and in parallel exposed to vehicle or LPS for 24 h. The concentrations of the following cytokines and chemokines were measured in the culture media by ELISA: (A, C) IL-12, and (B, D) IL-10. (E, F) Mice were administered with IFN-b or a vehicle 24 h PPI, and peritoneal exudates were collected 48 h PPI. (E) Equal volumes of cell-free fluids were run by SDS-PAGE, followed by immunoblotting for PR3; representative blot images. (F) Proteolytic activity of PR3 in exudates was examined by casein zymography; representative gel images are shown. Full blot and zymography images are shown in Figure S13. Data are representative of two to three independent experiments and are presented as mean ± SEM, n = 4–8. Statistical significance relative to vehicle-treated mice or between LPS-stimulated WT and Gal-1null macrophages was analyzed by two-tailed Student's t-test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The indicated reagents were obtained as follows: ELISA kits for mouse TNF-a, IL-10, IL-6, and IL-12 (Biolegend), and for mouse CCL2 and CCL5 (R&D systems); FITC-conjugated anti-mouse Gr-1, FITC-conjugated anti-mouse Ly6G, PE-conjugated antimouse F4/80, PerCP-conjugated anti-mouse CD11b, PBconjugated anti-mouse Ly6C and PE/Cy7-conjugated antimouse TLR4 (Biolegend); APC-conjugated anti-mouse Tim4 (Miltenyi); Alx647-conjugated anti-mouse IgG, goat antimouse arginase-1, and rabbit anti-IFN-b antibodies (Abcam); rabbit anti-mouse 12/15-lipoxygenase antibody (Cayman Chemical); goat anti-mouse CD11b, goat anti-mouse b-actin and goat anti-PR3 antibodies (Santa Cruz Biotechnology); antip-STAT-1 and anti-p-STAT3 (Cell Signalling); anti-goat and anti-rabbit horseradish peroxidase-conjugated antibodies (Jackson ImmunResearch laboratories).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot, Activity Assay, Zymography, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Endogenous SLPI contributes to the regulation of inflammatory responses in peritoneal macrophages by modulating MMP-9 production

doi: 10.3389/fimmu.2025.1563845

Figure Lengend Snippet: SLPI regulates MMP-9 secretion in resident peritoneal macrophages. (A) SLPI, IL-6 and TNF in supernatants of WT and Slpi -/- resident peritoneal macrophages incubated with LPS (100 ng/ml) for 24h.WT control vs WT LPS ** p< 0.01 by one-way ANOVA, Tuckey post hoc test. (B) IL-1α and IL-1β in supernatants of WT and Slpi -/- peritoneal macrophages incubated with LPS (100 ng/ml) for 3 h followed by addition of alum (100 µg/ml) or nigericin (10 µM). (C) Total cell and neutrophil count in WT and Slpi -/- mice injected i.p. with PBS or 100 μg alum for 24h. (D) Representative immunoblots of MMP-9 and SLPI in supernatants and lysates of WT and Slpi -/- peritoneal macrophages incubated with LPS (100 ng/ml) for 24h.Samples were resolved by SDS-PAGE and probed by Western blotting. β-actin was used as loading control. (E) Densitometry analysis of (D) WT LPS vs Slpi -/- LPS ***p < 0.001 by one-way ANOVA, Tukey post hoc test. (F) MMP-9 in supernatants of WT and Slpi -/- resident peritoneal macrophages incubated with LPS (100 ng/ml) for indicated times. WT LPS vs Slpi -/- LPS ***p < 0.001 by one-way ANOVA, Tukey post hoc test. (G) Representative zymography of equal volume of supernatants obtained from WT and Slpi -/- peritoneal macrophages incubated with LPS (100 ng/ml) for 24h.Representative of four separate experiments. (H) MMP-9 in supernatants of WT and Slpi -/- resident peritoneal macrophages incubated with DMSO or inhibitors: JNK-INH-8 (10 μM), SP600125 (20 μM), PD98059 (20 μM) or SB203580 (10 μM) for 1h followed by LPS treatment (100 ng/ml) for 24h.LPS vs LPS + inhibitor *p<0.05 by one-way ANOVA, Tukey post hoc test. (I) SLPI, IL-6 and MMP-9 in supernatants of WT and Slpi -/- resident peritoneal macrophages incubated with LPS (100 ng/ml) and IFN-γ (50 ng/ml) for 24h.Control vs IFN−γ *p<0.05, ***p<0.001 by multiple unpaired t-test. (A, B, F, H, I) Data are presented as the mean of three (A, B, F) or four (H, I) independent experiments. Error bars show means ± SEM. (C) Data represent 5 to 6 mice per experimental group pooled from two independent experiments. Error bars show means ± SEM.

Article Snippet: The following reagents were used: Alhydrogel (Cat. No. vac-alu-250) referred as “alum”, ultrapure LPS from E. coli serotype O55:B5 (Cat. No. Tlrl-pb5lps) for in vivo experiments, mouse TLR1–9 Agonist Kit (Cat. No. tlrl-kit1mw), nigericin (Cat. No. tlrl-nig) all from Invivogen (Toulouse, France); BD DIFCO fluid thioglycolate medium (Cat. No. 225650; BD, Argenta, Poznan, Poland); TMB substrate reagent set (Cat. No. 555214; BD, Warsaw, Poland), streptavidin PE (Cat. No. 554061; BD, Warsaw, Poland); LPS from E. coli serotype EH100 (Cat. No. ALX-581-010-L001; Enzo, Farmingdale, USA) for in vitro experiments; murine recombinant IFN-γ (Cat. No. 485-MI; R&D Systems, Bio-Techne, Warsaw, Poland); Peprotech murine recombinant IL-4 (Cat. No. 214-14), Peprotech murine recombinant M-CSF (Cat. No. 315-02), Gibco DMEM with high glucose (Cat. No. 11965092), penicillin-streptomycin (Cat. No. 15070063), Gibco heat-inactivated fetal bovine serum (Cat. No. 10500064), eBioscience Intracellular Fixation Buffer (Cat. No. 00-8222-49), eBioscience Foxp3/Transcription Factor Fixation/Permeabilization concentrate and diluent (Cat. No. 00-5521-00), eBioscience permeabilization buffer (10X) (Cat. No. 00-8333-56) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); inhibitors: JNK-IN-8 (Cat. No. HY-13319), SP600125 (Cat. No. HY-12041), PD98059 (Cat. No. HY-12028) and SB203580 (Cat. No. HY-10256) all from MedChemExpress (Sollentuna, Sweden); PBS (Cat. No. P04-36500; PAN-Biotech, Immuniq, Zory, Poland); sodium chloride injection solution (Cat. No. 3117301, Polpharma, Starogard Gdanski, Poland); BSA (Cat. No. ALB004; BioShop, Epro Science, Wladyslawowo, Poland); Tween-20 (Cat. No. P9416), saponin (Cat. No. 84510), gelatin type A from porcine skin (Cat. No. G-2625) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); Triton X-100 (Cat. No. 841810492; Poch SA, Gliwice, Poland); 2x Laemmli sample buffer (Cat. No. 1610737; Bio-Rad, Warsaw, Poland); WesternBright ECL HRP substrate (Cat. No. K-12045-D20, Advansta, San Jose, California, USA); paraformaldehyde (Cat. No. 43368.9M; VWR International, Gdansk, Poland); Zombie Aqua Fixable Viability Kit (Cat. No. 423102; Biolegend, Amsterdam, The Netherlands).

Techniques: Incubation, Control, Injection, Western Blot, SDS Page, Zymography

Journal: Biomedicines

Article Title: Factors Released by Polarized Neutrophil-like Cells Modulate Cardiac Fibroblast Phenotype and Limit the Inflammatory Response After Myocardial Infarction

doi: 10.3390/biomedicines13112829

Figure Lengend Snippet: Impact of cell culture conditions on human cardiac fibroblasts (CFs) morphology and phenotype. ( A ) Morphology of CFs after 48h in conventional 2D culture ( a , c ) and 3D culture on ARdH hydrogel ( b , d ), as determined by F-actin labeling with phalloidin (red) and DAPI nuclear staining (cyan). The scale bar indicates 200 µm for ( a , b ), and 50 µm for ( c , d ). ( B ) Cell viability of CFs after 48 h in 2D and 3D culture, as determined by MTT assay; ( C ) MCP-1 released in conditioned media by CFs cultured in 2D and 3D conditions determined by ELISA assay; ( D ) mRNA expression of α-SMA, MCP-1, and IL-1β in CFs isolated from 3D-hydrogel or 2D culture after 48 h. ( E ) α-SMA protein expression in CFs from aortic root-derived hydrogel (ARdH) or 2D culture, determined by Western Blot; n = 3, * p < 0.05. Data is represented as mean ± SEM.

Article Snippet: Secreted levels of human monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), macrophage inflammatory protein (MIP-1α, CCL3), IL-6, MMP-1, MMP-9, TGF-β1, and mouse S100A8/A9, MCP-1, IL-6, and TGF-β1 were measured in conditioned media/mice plasma using DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA, catalog codes: DY279 (MCP-1), DY201 (IL-1β), DY270 (MIP-1α), DY206 (IL-6), DY901 (MMP-1), DY911 (MMP-9), DY240 (TGF-β1) for human, and DY8596 (S100A8/A9), DY479 (MCP-1), DY406 (IL-6), and DY1679 (TGF-β1) for mouse), according to the manufacturer’s protocol.

Techniques: Cell Culture, Labeling, Staining, MTT Assay, Enzyme-linked Immunosorbent Assay, Expressing, Isolation, Derivative Assay, Western Blot

Journal: Biomedicines

Article Title: Factors Released by Polarized Neutrophil-like Cells Modulate Cardiac Fibroblast Phenotype and Limit the Inflammatory Response After Myocardial Infarction

doi: 10.3390/biomedicines13112829

Figure Lengend Snippet: Profile of inflammatory mediators and of matrix metalloproteinases (MMP) following a 24 h indirect co-culture of human CFs grown on ARdH hydrogel with control granulocytes (CfN), or their pro-inflammatory (N1) or anti-inflammatory (CfN2) subtypes. ( A ) mRNA level of inflammatory cytokines and chemokines in CFs grown on ARdH hydrogel and indirectly interacted with granulocytes subsets, determined by qPCR—normalized to B2M and represented as fold change compared with C (Cf only); ( B ) Levels of secreted inflammatory cytokines and chemokines in conditioned media from indirect co-cultures as evaluated by ELISA assay; ( C ) IL-1β protein level in CFs lysates following indirect interaction with neutrophil-like cell subtypes, as determined by Western Blot assay and normalized to β-actin; ( D ) Metalloproteases MMP-1, -2, -3, -9 and -13 mRNA expression in CFs indirectly co-cultured with neutrophil-like subtypes; ( E ) Concentration of soluble MMP-1 and MMP-9 quantified in conditioned media as evaluated by ELISA assay; ( F , G ) Protein expression of MMP-9/-1 in CFs following indirect interaction, as evaluated by Western Blot and normalized to β-actin; ( H ) The enzymatic activity of gelatinase MMP-9 in the conditioned media assessed by SDS-PAGE gelatine zymography; ( I ) Intracellular signaling: MAPKs (p38 and ERK1/2) and NF-κB phosphorylation in human CFs following indirect interaction with neutrophil-like cell subtypes; ( J ) Proliferative capacity of primary mouse CFs assessed using the xCelligence system: C+—fibroblasts exposed to DMEM/F12 with 10%FBS and C-—fibroblasts exposed to DMEM/F12 without FBS; N1/N2—fibroblasts exposed to mouse N1/N2 conditioned media; ( K ) Cell index after ~3 days of exposure to N1/N2 conditioned media. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001 Data is represented as mean ± SEM.

Article Snippet: Secreted levels of human monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), macrophage inflammatory protein (MIP-1α, CCL3), IL-6, MMP-1, MMP-9, TGF-β1, and mouse S100A8/A9, MCP-1, IL-6, and TGF-β1 were measured in conditioned media/mice plasma using DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA, catalog codes: DY279 (MCP-1), DY201 (IL-1β), DY270 (MIP-1α), DY206 (IL-6), DY901 (MMP-1), DY911 (MMP-9), DY240 (TGF-β1) for human, and DY8596 (S100A8/A9), DY479 (MCP-1), DY406 (IL-6), and DY1679 (TGF-β1) for mouse), according to the manufacturer’s protocol.

Techniques: Co-Culture Assay, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Cell Culture, Concentration Assay, Activity Assay, SDS Page, Zymography, Phospho-proteomics

Journal: Biomedicines

Article Title: Factors Released by Polarized Neutrophil-like Cells Modulate Cardiac Fibroblast Phenotype and Limit the Inflammatory Response After Myocardial Infarction

doi: 10.3390/biomedicines13112829

Figure Lengend Snippet: The impact of granulocyte subtypes on the expression of molecules associated with fibrosis in human CFs following 24 h indirect co-culture with neutrophil-like cell subsets. ( A ) mRNA of fibrotic markers in CFs grown on ARdH hydrogel and indirectly interacted with granulocyte subsets, determined by qPCR assay—normalized to β-actin and represented as fold change compared with control Cf (non-interacted); ( B ) Concentration of soluble TGF-β1 quantified in conditioned media following indirect interaction as evaluated by ELISA assay; n = 3, * p < 0.05, ** p < 0.01. Data is represented as mean ± SEM.

Article Snippet: Secreted levels of human monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), macrophage inflammatory protein (MIP-1α, CCL3), IL-6, MMP-1, MMP-9, TGF-β1, and mouse S100A8/A9, MCP-1, IL-6, and TGF-β1 were measured in conditioned media/mice plasma using DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA, catalog codes: DY279 (MCP-1), DY201 (IL-1β), DY270 (MIP-1α), DY206 (IL-6), DY901 (MMP-1), DY911 (MMP-9), DY240 (TGF-β1) for human, and DY8596 (S100A8/A9), DY479 (MCP-1), DY406 (IL-6), and DY1679 (TGF-β1) for mouse), according to the manufacturer’s protocol.

Techniques: Expressing, Co-Culture Assay, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Factors Released by Polarized Neutrophil-like Cells Modulate Cardiac Fibroblast Phenotype and Limit the Inflammatory Response After Myocardial Infarction

doi: 10.3390/biomedicines13112829

Figure Lengend Snippet: Effect of N1/N2 secretome (SN1/SN2) in modulating the inflammatory phase—day 1 post-MI. ( A , B ) Immunofluorescence of Ly6G (red) and MPO (red) in cardiac tissue from MI mice with or without treatment with SN1/SN2, 1-day post-MI. Nuclei are counterstained with DAPI (cyan). Scale bar always represents 100 μm. ( C ) Neutrophil elastase activity detected directly in explanted mouse hearts at 1-day post-MI; ( D – F ) Levels of IL-6, MCP-1, and TGF-β1, respectively, in plasma from MI mice treated or not with SN1/SN2, as evaluated by ELISA. n ≥ 3. Data is represented as mean ± SD.

Article Snippet: Secreted levels of human monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), macrophage inflammatory protein (MIP-1α, CCL3), IL-6, MMP-1, MMP-9, TGF-β1, and mouse S100A8/A9, MCP-1, IL-6, and TGF-β1 were measured in conditioned media/mice plasma using DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA, catalog codes: DY279 (MCP-1), DY201 (IL-1β), DY270 (MIP-1α), DY206 (IL-6), DY901 (MMP-1), DY911 (MMP-9), DY240 (TGF-β1) for human, and DY8596 (S100A8/A9), DY479 (MCP-1), DY406 (IL-6), and DY1679 (TGF-β1) for mouse), according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Activity Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Factors Released by Polarized Neutrophil-like Cells Modulate Cardiac Fibroblast Phenotype and Limit the Inflammatory Response After Myocardial Infarction

doi: 10.3390/biomedicines13112829

Figure Lengend Snippet: Effect of N1/N2 secretome (SN1/SN2) in the reparative phase—7 days post-MI. ( A ) Immunofluorescence staining of α-SMA (red) and MPO (red) in mouse cardiac tissue from MI mice with or without treatment with SN1/SN2. Nuclei are counterstained with DAPI (cyan). Scale bar always represents 100 μm; Signal intensity was quantified within the infarcted zone, selected from slide scans acquired at 20× magnification, and expressed as mean fluorescence intensity (MFI) per infarct area ( a1 , a2 ). ( B – D ) Levels of IL-6, MCP-1 and TGF-β1, respectively, in plasma from mice with MI, treated or not with SN1/SN2, as evaluated by ELISA assay. n ≥ 3, * p < 0.05, ** p < 0.01. Data is represented as mean ± SD.

Article Snippet: Secreted levels of human monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), macrophage inflammatory protein (MIP-1α, CCL3), IL-6, MMP-1, MMP-9, TGF-β1, and mouse S100A8/A9, MCP-1, IL-6, and TGF-β1 were measured in conditioned media/mice plasma using DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA, catalog codes: DY279 (MCP-1), DY201 (IL-1β), DY270 (MIP-1α), DY206 (IL-6), DY901 (MMP-1), DY911 (MMP-9), DY240 (TGF-β1) for human, and DY8596 (S100A8/A9), DY479 (MCP-1), DY406 (IL-6), and DY1679 (TGF-β1) for mouse), according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Staining, Fluorescence, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Matrix metalloproteinase promotes elastic fiber degradation in ligamentum flavum degeneration

doi: 10.1371/journal.pone.0200872

Figure Lengend Snippet: A: Representative gelatin zymography analysis of LF fibroblasts in response to IL-6/sIL-6Rα stimulation for 24 h with or without Stattic. B: Quantitative analysis of data shown in panel A (n = 3). C: Concentration of soluble elastin following incubation for 6 h with the supernatant of IL-6-stimulated cultures with or without Stattic (n = 3). Data represent the mean ± SEM. **P < 0.01.

Article Snippet: LF fibroblasts with or without 50 nM Stattic pretreatment were stimulated with IL-6 (300 ng/ml) for 24 h. MMP-2 and -9 activities in the culture supernatant were measured with an SDS-PAGE gelatin zymography kit (Cosmo Bio, Tokyo, Japan) according to the manufacturer’s instructions.

Techniques: Zymography, Concentration Assay, Incubation

Journal: Oncogene

Article Title: TrkB induces EMT and has a key role in invasion of head and neck squamous cell carcinoma.

doi: 10.1038/onc.2009.486

Figure Lengend Snippet: Figure 1. Tyrosine kinase B (TrkB) is over-expressed in human head and neck squamous cell carcinoma (HNSCC) tumors. (a) Complementary DNA (cDNA) microarray analysis revealed that messenger RNA (mRNA) expression of NTRK2 correlated with expression of BDNF in 71 previously untreated human tumors (P<0.005) (b) Expression of TrkB and brain-derived neurotrophic factor (BDNF) in representative samples on a human HNSCC tissue array. Tumors were analyzed and graded for TrkB (P<0.0001) and BDNF (P<0.001) expression (scale: 0–3). (c) Protein lysates from subconfluent HNSCC or normal cell lines were separated by SDS–PAGE and assessed with the indicated antibodies. Mouse brain (MsBr) was used as a positive control for TrkB expression. (d) Schematic representation of the TrkB receptor. Exon numbers indicate regions of the receptor that were assessed for genomic mutational analysis (see Table 1). TM, transmembrane domain; TK, tyrosine kinase.

Article Snippet: Short-hairpin RNA constructs targeting TrkB were purchased from Origene (Rockville, MD, USA) (TR320436) and were introduced into cells through retroviral infection according to the manufacturer’s protocol and were selected with puromycin (1 μg/ml).

Techniques: Microarray, Expressing, Derivative Assay, SDS Page, Positive Control

Journal: Oncogene

Article Title: TrkB induces EMT and has a key role in invasion of head and neck squamous cell carcinoma.

doi: 10.1038/onc.2009.486

Figure Lengend Snippet: Figure 2. Tyrosine kinase B (TrkB) expression is associated with differential chemotactic responsiveness to brain-derived neurotrophic factor (BDNF). (a) Head and neck squamous cell carcinoma (HNSCC) cell lines were exposed to BDNF (100 ng/ml) in Transwell migration plates and assessed for chemotactic cellular migration after 24 h. Cells were counted in five high-powered fields, analyzed for differential migration with ImagePro. Epidermal growth factor (EGF) (100 ng/ml) was added to a separate well as a positive control for migration experiments (data not shown). Columns, relative migration of cells in the presence or absence of BDNF; bars, s.e.m; NS, not significant. Representative experimental results from triplicate repeats. Magnification: ×100. (b) HNSCC cell lines were exposed to BDNF in Matrigel invasion plates and assessed for cellular invasion after 24 h. Cells were counted in five high-powered fields and analyzed for differential migration with ImagePro. EGF was added to a separate well as a positive control for invasion experiments (data not shown). Columns, relative invasion of cells in the presence or absence of BDNF; bars, s.e.m; NS, not significant. Magnification: ×100. (c) BDNF induces matrix metallopeptidase 9 (MMP-9) expression and activation in HNSCC. Quantitative RT–PCR (left) and gelatin zymography (right) were performed to determine the induction of MMP expression and function under the control of BDNF stimulation. (d) Cell lines were exposed to BDNF (100 ng/ml) with or without triciribine (5 μM) in migration plates and assessed for migration after 24 h (left). Cells were counted in five high-powered fields and analyzed for

Article Snippet: Short-hairpin RNA constructs targeting TrkB were purchased from Origene (Rockville, MD, USA) (TR320436) and were introduced into cells through retroviral infection according to the manufacturer’s protocol and were selected with puromycin (1 μg/ml).

Techniques: Expressing, Derivative Assay, Migration, Positive Control, Activation Assay, Quantitative RT-PCR, Zymography, Control

Journal: Oncogene

Article Title: TrkB induces EMT and has a key role in invasion of head and neck squamous cell carcinoma.

doi: 10.1038/onc.2009.486

Figure Lengend Snippet: Figure 3. Transient manipulation of tyrosine kinase B (TrkB) in head and neck squamous cell carcinoma (HNSCC) alters ligand-mediated migration and invasion. (a) OSC19 cells were transiently transfected with siRNA constructs, and lysates from subconfluent cells were analyzed by SDS–PAGE with the indicated antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) knockdown was assayed as a positive control. (b) OSC19 cells were transiently transfected with small interfering RNA (siRNA) against TrkB (construct 3) or a non-targeted siRNA (inset), and seeded on Transwell migration plates for 24 h in the presence or absence of BDNF (100 ng/ml). Cells were counted in five high-powered fields and analyzed for differential migration with ImagePro. Experiments were performed in triplicate and repeated three times. Columns, number of migrated cells in the presence or

Article Snippet: Short-hairpin RNA constructs targeting TrkB were purchased from Origene (Rockville, MD, USA) (TR320436) and were introduced into cells through retroviral infection according to the manufacturer’s protocol and were selected with puromycin (1 μg/ml).

Techniques: Migration, Transfection, Construct, SDS Page, Knockdown, Positive Control, Small Interfering RNA

Journal: Oncogene

Article Title: TrkB induces EMT and has a key role in invasion of head and neck squamous cell carcinoma.

doi: 10.1038/onc.2009.486

Figure Lengend Snippet: Figure 4. Morphological and functional alterations induced by targeted reduction of tyrosine kinase B (TrkB). (a) Messenger RNA (mRNA) (top) and protein (bottom) levels of TrkB after stable transfection of OSC19-Luc cells with vectors targeting an irrelevant sequence (shRNA-NT) or TrkB (shRNA-TrkB). Quantification with ImagePro was performed to determine the relative change in TrkB protein expression among the various constructs (right). (b) Cellular morphology of stably transfected cells ( ×200, inset ×40) under both light (top) and fluorescence (bottom) microscopy. Deregulation of epithelial-to-mesenchymal transition (EMT) markers in transfected cells was determined by 10% SDS–PAGE and assayed with the indicated antibodies (right). (c) Constitutive migration (left) and invasion (right) of parental (pRS), shRNA-NT or shRNA-TrkB transfected cells toward a 10% fetal bovine serum (FBS) gradient. Experiments were performed in triplicate and repeated three times. Columns, relative migration or invasion of cells; bars, s.e.m.; magnification: ± 100. (d) brain-derived neurotrophic factor (BDNF)-mediated chemotactic invasion of shRNA-NT

Article Snippet: Short-hairpin RNA constructs targeting TrkB were purchased from Origene (Rockville, MD, USA) (TR320436) and were introduced into cells through retroviral infection according to the manufacturer’s protocol and were selected with puromycin (1 μg/ml).

Techniques: Functional Assay, Stable Transfection, Sequencing, shRNA, Expressing, Construct, Transfection, Fluorescence, Microscopy, SDS Page, Migration, Derivative Assay

Journal: Oncogene

Article Title: TrkB induces EMT and has a key role in invasion of head and neck squamous cell carcinoma.

doi: 10.1038/onc.2009.486

Figure Lengend Snippet: Figure 5. Tyrosine kinase B (TrkB)-mediated cellular invasion is AKT dependant. (a) Differential phosphorylation of AKT was determined with 10% SDS–PAGE. (b) Short hairpin RNA (shRNA)-transfected OSC19-Luc cells were seeded onto Matrigel-coated wells, with 10% fetal bovine serum (FBS) with or without triciribine (5 μM) in the lower chamber. Experiments were performed in triplicate and repeated three times. Columns, relative invasion of cells (expressed as a ratio of invaded cellsTCN/invaded cellscontrol); bars, s.e.m.; statistical significance was determined using Student’s t-test. Magnification: × 100. (c) Brain-derived neurotrophic factor (BDNF)-mediated chemotactic invasion of shRNA-NT (left) and shRNA-TrkB (right) cells was determined in the presence or absence of triciribine (TCN), as above. Columns, relative invasion of cells; bars, s.e.m.; magnification: × 100. Experiments were performed in triplicate and repeated three times. (d), TCN abrogates BDNF-mediated AKT phospharylation. Serum-starved shRNA-NT or shRNA-TrkB cells

Article Snippet: Short-hairpin RNA constructs targeting TrkB were purchased from Origene (Rockville, MD, USA) (TR320436) and were introduced into cells through retroviral infection according to the manufacturer’s protocol and were selected with puromycin (1 μg/ml).

Techniques: Phospho-proteomics, SDS Page, shRNA, Transfection, Derivative Assay

Journal: Oncogene

Article Title: TrkB induces EMT and has a key role in invasion of head and neck squamous cell carcinoma.

doi: 10.1038/onc.2009.486

Figure Lengend Snippet: Figure 6. Downregulation of tyrosine kinase B (TrkB) suppresses tumor growth in an orthotopic model of head and neck squamous cell carcinoma (HNSCC). (a) In vivo bioluminescence reveals suppression of tumor growth in orthotopically implanted cells harboring short hairpin RNA (shRNA) targeting TrkB (OSC19-shRNA-TrkB; n = 8) or a non-targeting construct (OSC19-shRNA-NT; n = 9). Results are representative of three independent experiments. (b) Tumor size is inhibited by 62% in tumors with TrkB knockdown. (c), Hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) analysis of tumors. IHC confirmed knockdown of TrkB in the shRNA-TrkB primary tumors compared with shRNA-NT tumors ( ×200, inset: ×50). Alterations in E-cadherin, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling), Ki67 and proliferating cell nuclear antigen (PCNA) were confirmed by IHC. NT, non targeting.

Article Snippet: Short-hairpin RNA constructs targeting TrkB were purchased from Origene (Rockville, MD, USA) (TR320436) and were introduced into cells through retroviral infection according to the manufacturer’s protocol and were selected with puromycin (1 μg/ml).

Techniques: In Vivo, shRNA, Construct, Knockdown, Staining, Immunohistochemical staining, TUNEL Assay

Journal: Oncogene

Article Title: TrkB induces EMT and has a key role in invasion of head and neck squamous cell carcinoma.

doi: 10.1038/onc.2009.486

Figure Lengend Snippet: Figure 7. Tyrosine kinase B (TrkB) overexpression induces epithelial-to-mesenchymal transition (EMT) and head and neck squamous cell carcinoma (HNSCC) migration and invasion. (a) Tu138 cells were infected with a lentiviral vector containing full-length TrkB, grown to confluency, and analyzed by 7% SDS–PAGE with the indicated antibodies. (b) Morphological alterations of Tu138 cells after stable infection with either an empty vector or TrkB-containing vector in 10% fetal bovine serum (FBS; magnification: × 200). (c) Mock or TrkB-transfected cells were seeded onto Transwell migration plates in the presence of either BDNF or epidermal growth factor (EGF) (positive control) and then analyzed for migration after 24 h. Experiments were performed in triplicate and repeated three times. Columns, relative migration of cells; bars, s.e.m; magnification: × 100. (d) Mock or TrkB-infected cells were seeded onto 6-well plates, grown to confluency and assessed for haptotaxis after a scratch wound was made. Images were taken at 0 and 8h, and degree of wound closure was determined with ImagePro. Experiments were performed in triplicate and repeated three times. Columns, percentage of wound closure from 0 h; bars, s.e.m; magnification ×100. (e) Expression of TrkB positively correlated with N-cadherin (P<0.0005) but negatively correlated with E-cadherin (P<0.01) in human HNSCC tumors. (f) Hypothetical model of the role of TrkB in HNSCC pathobiology.

Article Snippet: Short-hairpin RNA constructs targeting TrkB were purchased from Origene (Rockville, MD, USA) (TR320436) and were introduced into cells through retroviral infection according to the manufacturer’s protocol and were selected with puromycin (1 μg/ml).

Techniques: Over Expression, Migration, Infection, Plasmid Preparation, SDS Page, Transfection, Positive Control, Expressing

Journal: The American Journal of Pathology

Article Title: Matrix Metalloproteinase-1 Produced by Human CXCL12-Stimulated Natural Killer Cells

doi: 10.2353/ajpath.2006.050676

Figure Lengend Snippet: Expression of MMPs from CXCL12-stimulated NK cells. NK cells were incubated in serum-free RPMI 1640 containing 500 U/ml IL-2 or 20 ng/ml CXCL12 for 24 hours, and the conditioned medium and total cell lysates were prepared as described in Materials and Methods. A: The concentrated conditioned medium was separated on SDS-PAGE and blotted with anti-MMP-1 antibody and visualized with Super Signal West Pico chemiluminescent substrate. Molecular markers (kd) are shown in the left column. Total RNA was isolated from NK cells cultured in the presence of CXCL12 for 0, 2, 4, or 6 hours and subjected to RT-PCR experiments by using specific primers for MMP-1 (top) or GAPDH (bottom). The RT-PCR results were subjected to densitometry analysis as described in Materials and Methods. The result is shown as the ratio of MMP-1 to GAPDH at each time point. B: The concentrated conditioned medium was analyzed by gelatin-zymography as described in Materials and Methods. As a standard, conditioned medium prepared from HT1080 cells stimulated with ConA was used to localize pro- (top), intermediate (middle), and active (bottom) forms of MMP-2 are shown as lines. Molecular markers (kd) are shown in the left column. C: Total cell lysates were separated on SDS-PAGE, blotted with anti-MMP-14 antibody, and visualized with Super Signal West Pico chemiluminescent substrate. As a standard, HT1080 cells lysates stimulated with ConA were used. Note that the MT1-MMP proteins were detected as three species. The 56- and 54-kd bands represent zymogen and active form, respectively.47 The 45-kd band is a degraded MT1-MMP.47 Molecular markers (kd) are shown in the left column.

Article Snippet: Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Total RNA was isolated from human NK cells by RNeasy kit (Qiagen, Valencia, CA).

Techniques: Expressing, Incubation, SDS Page, Isolation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Zymography

Journal: Oncogene

Article Title: Involvement of the SKP2-p27(KIP1) pathway in suppression of cancer cell proliferation by RECK.

doi: 10.1038/onc.2011.570

Figure Lengend Snippet: Figure 2 Downregulation of SKP2 after RECK expression in two human tumor cell lines. (a) Normalized GeneChip data for the major transcripts of SKP2, p21 (CDKN1A), p27 (CDKN1B) and p57 (CDKN1C) expressed in SW620 cells at 48 h after infection with Ad-Z (gray bar) or Ad-RECK (black bar). (b) Detection of RECK, SKP2 and p27 transcripts by semi-quantitative RT–PCR. Total RNA from SW620 cells at 48 h after mock treatment (m) or infection with Ad-Z (Z) or Ad-RECK (R) (lanes 1–3) and total RNA from HT1080 cells stably transfected with a control vector (V) or the vector expressing RECK (R) (lanes 4 and 5) were analyzed. HPRT1 was used as a loading control. (c) Immunoblot detection of the RECK, SKP2 and p27 proteins in whole-cell lysates of SW620 at 48 h after mock treatment (lane 1) or after infection with Ad-Z (lane 2) or Ad-RECK (lane 3). GAPDH was used as a loading control. (d) Immunoblot detection of RECK, SKP2 and p27 proteins in whole-cell lysates of HT1080 cells stably transfected with a control vector (lane 1) or the vector expressing RECK (lane 2). (e) Growth curves of HT1080 cells stably transfected with a control vector (open squares) or a vector expressing RECK (filled squares). Bar represents mean±s.d. (n ¼ 3). (f) Effects of RECK knockdown on the levels of SKP2 and p27. RECK-transfected HT1080 cells plated on the previous day were incubated in growth medium (lane 2) or treated with vehicle (lane 1), a control siRNA (lane 3) or an siRNA targeting RECK (lane 4) for 48 h. Whole-cell lysates were subjected to immunoblot assay. Experiments were repeated three times with similar results. (g) Morphology of NIH3T3 cells plated at two different cell densities (3 104 (low) and 2.5 105 (high) per well in six-well plate) and incubated for 48 h. Scale bar: 50 mm. (h) Effects of cell density on the levels of Reck, Skp2 and p27 proteins in NIH3T3 cells. Lysates of the cells shown in (g) were subjected to immunoblot assay. GAPDH was used as a loading control. (i) Effects of conditional Reck-knockout on the levels of Skp2 and p27 in MEFs. R1B2-6 cells incubated in the absence or presence of 250 nM 4-hydroxytamoxifen for 96 h and then in GM for 48 h were subjected to immunoblot assay.

Article Snippet: Proteins in the cell lysates prepared, as described previously (Oh et al., 2001), were quantified using the DC protein assay kit (Bio-Rad, Hercules, CA, USA), separated by SDS–PAGE (10% acrylamide) and subjected to indirect immunoblot assay using the following antibodies as primary antibodies (antigen (source)): RECK (5B11D12) (Takahashi et al., 1998), p27 (BD Biosciences, Pharmingen, San Jose, CA, USA), SKP2 (Cell Signaling Technology, Danvers, MA, USA), and GAPDH (6C5, Ambion, Carlsbad, CA, USA).

Techniques: Expressing, Infection, Quantitative RT-PCR, Stable Transfection, Transfection, Control, Plasmid Preparation, Western Blot, Knockdown, Incubation, Knock-Out

Journal: Oncogene

Article Title: Involvement of the SKP2-p27(KIP1) pathway in suppression of cancer cell proliferation by RECK.

doi: 10.1038/onc.2011.570

Figure Lengend Snippet: Figure 3 Involvement of p27 in Ad-RECK-induced growth arrest and cellular senescence. (a) Effects of p27 siRNA on the levels of RECK, SKP2 and p27 in SW620 cells after infection with Ad-RECK. SW620 cells pretreated for 24 h with vehicle (lane 1), a control siRNA (lane 2) or an siRNA targeting p27 (lane 3) were infected with Ad-RECK and incubated for 72 h. Whole-cell lysates were subjected to immunoblot assay. (b) Effects of p27 siRNA on the growth of SW620 cells after infection with Ad-Z or Ad-RECK. SW620 cells pretreated for 24 h with either vehicle (panels 1–3), the control siRNA (panels 4–6) or the p27 siRNA (panels 7–9) were left uninfected (mock; panels 1, 4 and 7) or infected with either Ad-Z (panels 2, 5 and 8) or Ad-RECK (panels 3, 6 and 9) and incubated for 72 h. Scale bar: 100 mm. (c) Effects of p27 siRNA on Ad-RECK-induced reduction in cell number. Cells treated as described in b were counted. The data are presented as the ratios to the vehicle-treated controls. Bar represents mean±s.e.m. (n ¼ 3). (d) Effects of p27 siRNA on Ad-RECK-induced activation of senescence-associated b-galactosidase. Cells treated as described in a were subjected to X-gal staining at pH 6. Scale bar: 50 mm.

Article Snippet: Proteins in the cell lysates prepared, as described previously (Oh et al., 2001), were quantified using the DC protein assay kit (Bio-Rad, Hercules, CA, USA), separated by SDS–PAGE (10% acrylamide) and subjected to indirect immunoblot assay using the following antibodies as primary antibodies (antigen (source)): RECK (5B11D12) (Takahashi et al., 1998), p27 (BD Biosciences, Pharmingen, San Jose, CA, USA), SKP2 (Cell Signaling Technology, Danvers, MA, USA), and GAPDH (6C5, Ambion, Carlsbad, CA, USA).

Techniques: Infection, Control, Incubation, Western Blot, Activation Assay, Staining

Journal: Oncogene

Article Title: Involvement of the SKP2-p27(KIP1) pathway in suppression of cancer cell proliferation by RECK.

doi: 10.1038/onc.2011.570

Figure Lengend Snippet: Figure 4 Effects of type I collagen on SKP2 expression. (a) Effects of type I collagen on SKP2 and p27 expression. SW620 cells plated on a regular dish (lane 1) or a dish coated with type I collagen (lane 2) were incubated for 48 h, and their lysates were subjected to immunoblot assay to estimate the levels of the indicated proteins. (b) Effects of fibronectin (FN) on SKP2 expression. SW620 cells plated on a regular dish (lane 1) or a dish coated with FN (lane 2) were incubated for 48 h, and their lysates were subjected to immunoblot assay to estimate the levels of SKP2 and the loading control GAPDH. (c) Collagenolytic activity of SW620 cells left uninfected (mock) or infected with Ad-Z or Ad-RECK as visualized by in situ zymography using a quenched fluorescent substrate, DQ type I collagen. Typical fluorescence/phase-contrast images of a relatively large cluster (row 1), small cluster (row 2) and single cell (row 3) are shown. Scale bar: 20 mm. (d–h) Effects of signaling inhibitors on collagen-mediated SKP2 downregulation in SW620 cells. Cells plated on regular (non) or collagen-coated (Col) dishes were treated for 48 h with either PP2 (a Src inhibitor, d), GF109203X (a PKC inhibitor, e) or Toxin B (a Rho/Rac/Cdc42 inhibitor, f) at the indicated doses and subjected to immunoblot assay. (g and h) Effects of signaling inhibitors on Ad-RECK-mediated SKP2 downregulation in SW620 cells. Cells were mock-treated (m) or infected with Ad-Z (Z) or Ad-RECK (R), treated for 48 h with GF109203X (g) or Toxin B (h) at the indicated dose and subjected to immunoblot assay.

Article Snippet: Proteins in the cell lysates prepared, as described previously (Oh et al., 2001), were quantified using the DC protein assay kit (Bio-Rad, Hercules, CA, USA), separated by SDS–PAGE (10% acrylamide) and subjected to indirect immunoblot assay using the following antibodies as primary antibodies (antigen (source)): RECK (5B11D12) (Takahashi et al., 1998), p27 (BD Biosciences, Pharmingen, San Jose, CA, USA), SKP2 (Cell Signaling Technology, Danvers, MA, USA), and GAPDH (6C5, Ambion, Carlsbad, CA, USA).

Techniques: Expressing, Incubation, Western Blot, Control, Activity Assay, Infection, In Situ, Zymography

Journal: Oncogene

Article Title: Involvement of the SKP2-p27(KIP1) pathway in suppression of cancer cell proliferation by RECK.

doi: 10.1038/onc.2011.570

Figure Lengend Snippet: Figure 6 A model consistent with our findings. RECK protects collagen from proteolytic degradation, and collagen downregulates SKP2 (possibly through integrin signaling), leading to decreased p27-degradation and consequent suppression of proliferation. Downregulation of RECK gene expression by growth factor (GF) as well as oncogenic signals have been found in previous studies (Takahashi et al., 1998; Sasahara et al., 1999; Hatta et al., 2009). There may be other pathways by which RECK suppresses cancer cell proliferation.

Article Snippet: Proteins in the cell lysates prepared, as described previously (Oh et al., 2001), were quantified using the DC protein assay kit (Bio-Rad, Hercules, CA, USA), separated by SDS–PAGE (10% acrylamide) and subjected to indirect immunoblot assay using the following antibodies as primary antibodies (antigen (source)): RECK (5B11D12) (Takahashi et al., 1998), p27 (BD Biosciences, Pharmingen, San Jose, CA, USA), SKP2 (Cell Signaling Technology, Danvers, MA, USA), and GAPDH (6C5, Ambion, Carlsbad, CA, USA).

Techniques: Gene Expression