Journal: BMC Cancer

Article Title: Extracellular Hsp90 and TGFβ regulate adhesion, migration and anchorage independent growth in a paired colon cancer cell line model

doi: 10.1186/s12885-017-3190-z

Figure Lengend Snippet: Investigating the status of the canonical TGF-β pathway and levels of Hsp90 in the genetically paired SW480 primary and SW620 secondary tumour-derived colon cancer cell lines. a The secretion of TGF-β1 by SW480 and SW620 cells was determined by means of an ELISA to quantify the levels of extracellular TGF-β1 in spent culture medium for a given cell number after 12 h incubation. Data represent the average of three independent experiments performed in triplicate and error bars indicate the standard error in the mean. b The levels of TGF-βRII receptor on the cell surface was determined by flow cytometry using a fluorescein isothiocyanate-conjugated antibody. Data was analysed using FlowJo software and gating carried out according to the IgG1 isotype control. Histograms show a shift in fluorescence for each sample (black line, unshaded) compared to the isotype control (grey shading) and the percentage of positive events is indicated for each sample. Data are representative of three independent experiments showing consistent results. c Intracellular levels of TGF-β1 in SW480 and SW620 cells were determined by (i) western blot analysis and compared to a histone loading control. The images are cropped from the full length western blot provided as Additional file . (ii) The normalized levels of pre-pro TGFβ1 from the western blot in (i) were determined by densitometry using ImageJ and calculated according the histone loading control. AU: arbitrary units. The positive control represents commercially obtained recombinant human TGFβ1 in its active form. d Activation of the TGF-β canonical pathway was determined by the phosphorylation and nuclear localization of Smad2/3. SW480 and SW620 cells were stained for pSMAD2/3 and nuclei were stained with Hoescht-33342. Immunofluorescence was detected using the Zeiss LSM 510 Meta laser scanning confocal microscope and the images analysed using Axiovision LE 4.7.1 software (Zeiss). (i) The first column shows the level of pSmad2/3 staining in the cells, pseudocoloured to white, while the second column shows a merged image of the nucleus pseudo-coloured to red and the pSMAD2/3 staining pseudocoloured to green. The third column shows frequency scattergrams obtained using colocalisation analysis on Image J,. Scale bars represent 20 μm. (ii) Graphs generated from profiles of the nucleus and pSMAD2/3 within individual cells analysed using Zen Lite Software showing the proportion of total pSMAD2/3 that is found in the nucleus. In all cases, analyses were performed on triplicate images containing at least 10 cells per image e The secretion of Hsp90β by SW480 and SW620 cells was quantified by means of an ELISA on spent culture medium after 12 h incubation. The data are representative of two individual experiments performed in triplicate and showing consistent results. f Whole cell lysates from SW480 and SW620 cells were analysed for Hsp90α and Hsp90β expression using western blot analysis. Where relevant, data was analysed using GraphPad Prism 4.03 software with errors bars indicating the standard error in the mean and a students t -test was performed to assess statistical significance. (* p < 0.05, ** p < 0.01)

Article Snippet: Recombinant native endotoxin-free human Hsp90β protein (SPR-102C) was from StressMarq Biosciences Inc., while recombinant endotoxin-free human TGF-β1 (carrier-free) (580704) was from BioLegend and bovine serum albumin (BSA) (10735078001) was from Roche.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry, Software, Fluorescence, Western Blot, Positive Control, Recombinant, Activation Assay, Staining, Immunofluorescence, Microscopy, Generated, Expressing

Journal: BMC Cancer

Article Title: Extracellular Hsp90 and TGFβ regulate adhesion, migration and anchorage independent growth in a paired colon cancer cell line model

doi: 10.1186/s12885-017-3190-z

Figure Lengend Snippet: The effect of addition or inhibition of TGF-β and Hsp90 on adhesion in the paired SW480 and SW620 colon cancer cell lines. SW480 a and SW620 b cells were treated with 2 ng/ml TGF-β1, 20 ng/ml Hsp90β, 100 nM SB431542, 100 μM novobiocin and 10 μg/ml αvβ6 integrin blocking antibody (anti- αvβ6) singly or in combinations as indicated on the x-axis. Absorbance at 590 nm of crystal violet stained adherent cells was normalized to the untreated control, given as 100%, and depicted by the solid horizontal line in the graph, showing the changes in cell adhesion for each treatment. All graphs are representative of the average of three independent experiments performed in triplicate and error bars indicate the standard error in the mean. Statistical analysis was performed using GraphPad Prism 4.03 software. A two-way analysis of variance (ANOVA) with Bonferroni post-test was performed and significance between untreated cells and those after each treatment (indicated by asterisks) and between particular treatments (indicated by hashes) are shown (* p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.05, ### p < 0.001, ns – not significant)

Article Snippet: Recombinant native endotoxin-free human Hsp90β protein (SPR-102C) was from StressMarq Biosciences Inc., while recombinant endotoxin-free human TGF-β1 (carrier-free) (580704) was from BioLegend and bovine serum albumin (BSA) (10735078001) was from Roche.

Techniques: Inhibition, Blocking Assay, Staining, Software

Journal: BMC Cancer

Article Title: Extracellular Hsp90 and TGFβ regulate adhesion, migration and anchorage independent growth in a paired colon cancer cell line model

doi: 10.1186/s12885-017-3190-z

Figure Lengend Snippet: The effect of addition or inhibition of TGF-β and Hsp90 on migration of the paired SW480 and SW620 colon cancer cell lines. SW480 a and SW620 b cells were treated with 2 ng/ml TGF-β1, 20 ng/ml Hsp90β, 100 nM SB431542, 100 μM novobiocin and 10 μg/ml αvβ6 integrin blocking antibody (anti- αvβ6) singly or in combinations as indicated on the x-axis and the effect of such treatment on migration assessed. All graphs are representative of the average of three independent experiments performed in triplicate and error bars indicate the standard error in the mean. Statistical analysis was performed using GraphPad Prism 4.03 software. A two-way analysis of variance (ANOVA) with Bonferroni post-test was performed and significance between untreated cells and those after each treatment (indicated by asterisks) and between particular treatments (indicated by hashes) are shown (* p < 0.05, ** p < 0.01, *** p < 0.001, ## p < 0.01, ### p < 0.001, ns – not significant)

Article Snippet: Recombinant native endotoxin-free human Hsp90β protein (SPR-102C) was from StressMarq Biosciences Inc., while recombinant endotoxin-free human TGF-β1 (carrier-free) (580704) was from BioLegend and bovine serum albumin (BSA) (10735078001) was from Roche.

Techniques: Inhibition, Migration, Blocking Assay, Software

Journal: BMC Cancer

Article Title: Extracellular Hsp90 and TGFβ regulate adhesion, migration and anchorage independent growth in a paired colon cancer cell line model

doi: 10.1186/s12885-017-3190-z

Figure Lengend Snippet: Investigation of the role of TGF-β and Hsp90 in anchorage-independent growth of SW480 and SW620 cells. a Photograph of tumourspheres formed by SW480 (i) and SW620 (ii) cells taken under a light microscope at 100x magnification. Scale bars indicate 100 μm. b Comparison of TGF-β1 and Hsp90β secretion by SW480 primary and SW620 secondary tumour-derived cells grown adherently and in suspension using a DuoSet ELISA kit (R and D systems) and sandwich ELISA method, respectively. Data shown are representative of three individual experiments carried out in triplicate and showing consistent results. Statistical significance was assessed using GraphPad Prism 4.03 software by means of a two-way analysis of variance (ANOVA) with Bonferroni post-test where n = 3. Comparisons in terms of the levels of proteins between adherent cells and tumoursphere (within cell lines) is indicated by asterisks (*), while comparisons between cell lines is indicated by hashes (#). c and d Analysis of the effect of addition or inhibition of TGF-β or Hsp90 on tumoursphere formation. Sphere forming efficiency (percentage of the total number of cells seeded that are able to form tumourspheres after 7 days) of SW480 c and SW620 cells d was normalized to that of untreated cells for each cell line (taken as 100%). Error bars indicate the standard error in the mean where n = 4. Statistical significance was assessed using GraphPad Prism 4.03 software by means of a two-way analysis of variance (ANOVA) with Bonferroni post-test and significance between untreated cells and those after each treatment (indicated by asterisks) as well as between particular treatments (indicated by hashes) are shown (* p < 0.05, ** p < 0.01, # p < 0.05, ## p < 0.01, ### p < 0.001)

Article Snippet: Recombinant native endotoxin-free human Hsp90β protein (SPR-102C) was from StressMarq Biosciences Inc., while recombinant endotoxin-free human TGF-β1 (carrier-free) (580704) was from BioLegend and bovine serum albumin (BSA) (10735078001) was from Roche.

Techniques: Light Microscopy, Derivative Assay, Enzyme-linked Immunosorbent Assay, Sandwich ELISA, Software, Inhibition

Journal: BMC Cancer

Article Title: Extracellular Hsp90 and TGFβ regulate adhesion, migration and anchorage independent growth in a paired colon cancer cell line model

doi: 10.1186/s12885-017-3190-z

Figure Lengend Snippet: Effect of pre-treatment of paired colon cancer cell lines by the addition or inhibition of TGF-β or Hsp90 under anchorage independent conditions on subsequent sensitivity to colon chemotherapeutics. SW480 (i) and SW620 (ii) cells were treated with either 2 ng/ml TGF-β1, 20 ng/ml Hsp90β, 100 nM SB431542 or 100 μM novobiocin upon seeding in a tumoursphere assay. After 7 days, tumourspheres were reseeded into regular adherent growth conditions and treated with 75 μM 5-fluorouracil a or 550 μM oxaliplatin b for 72 h. Cell viability was assessed compared to an untreated control for each pre-treatment using a MTT Cell Proliferation kit. Data are representative of two individual experiments carried out in triplicate and showing consistent results. Statistical significance was assessed by means of a two-way analysis of variance (ANOVA) with Bonferroni post-tests relative to the non-pre-treated control using GraphPad prism where n = 3 (* p < 0.05, ** p < 0.01)

Article Snippet: Recombinant native endotoxin-free human Hsp90β protein (SPR-102C) was from StressMarq Biosciences Inc., while recombinant endotoxin-free human TGF-β1 (carrier-free) (580704) was from BioLegend and bovine serum albumin (BSA) (10735078001) was from Roche.

Techniques: Inhibition, MTT Cell Proliferation

Journal: BMC Cancer

Article Title: Extracellular Hsp90 and TGFβ regulate adhesion, migration and anchorage independent growth in a paired colon cancer cell line model

doi: 10.1186/s12885-017-3190-z

Figure Lengend Snippet: Model describing the effect of a synergy between extracellular TGF-β1 and Hsp90β on downstream signalling in colon cancer cells. a In the presence of TGF-β1 alone, binding to the heterotetrameric TGF-βRI/II receptor complex triggers phosphorylation of Smad2/3 and subsequent activation of Smad4, whereupon the complex moves into the nucleus and triggers transcription of canonical Smad-responsive genes such as VEGF (tumour promoting) and Fas (tumour suppressing). b In the presence of both TGF-β1 and Hsp90β in the extracellular space, when binding of TGF-β1 to the TGF-βRI/II receptor complex is inhibited using SB431542, TGF-β1 and Hsp90β instead bind to αvβ6 integrin, triggering as yet unknown downstream non-Smad signalling pathways, culminating in the transcription of genes which promote metastatic behaviours including migration and anchorage-independent growth (AIG). This alternate pathway does not require inhibition of TGF-βRI/II and is constitutively active in SW620 cells, which represent a later stage of colon cancer compared to SW480 cells

Article Snippet: Recombinant native endotoxin-free human Hsp90β protein (SPR-102C) was from StressMarq Biosciences Inc., while recombinant endotoxin-free human TGF-β1 (carrier-free) (580704) was from BioLegend and bovine serum albumin (BSA) (10735078001) was from Roche.

Techniques: Binding Assay, Activation Assay, Migration, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *

doi: 10.1074/jbc.M113.494542

Figure Lengend Snippet: Siglec-15 and DAP12 form a complex in osteoclasts, and Siglec-15 clustering induces DAP12 phosphorylation and Akt signaling. A, co-immunoprecipitation (Co-IP) of DAP12 and Siglec-15. Protein lysates were prepared from non-differentiated RAW264.7 cells (control RAW) or RAW264.7-derived osteoclasts. Siglec-15 (left panels) and DAP12 (middle panels) immunoprecipitates as well as total lysates (right panels) were analyzed by Western blotting with Siglec-15 and DAP12 antibodies. The no lysate IPs were performed using fresh lysis buffer. B, analysis of cell signaling induced by Siglec-15 clustering. Control (C) or differentiated (D) RAW264.7 cells were treated with primary antibody (anti-Siglec-15 or control human IgG) at 4 °C followed by a secondary cross-linking antibody for the indicated times at 37 °C. Total lysates were analyzed by Western blotting with the indicated antibodies. C, DAP12 phosphorylation following Siglec-15 cross-linking. RAW264.7 cells were treated as in B for 5 min with secondary antibody (lanes 1, 2, and 5). As additional controls, some cells were lysed immediately after primary antibody incubation (lane 3) and for others, the secondary antibody was omitted from the 37 °C incubation media (lane 4). Protein extracts were prepared, and DAP12 immunoprecipitates were analyzed by blotting with anti-phosphotyrosine, anti-DAP12, and anti-Siglec-15 antibodies.

Article Snippet: Fab Preparation and Analysis Fab fragments were obtained from antibody B02 by ficin digest using the Mouse IgG1 Fab and F(ab′) 2 Preparation Kit (Pierce), according the manufacturer's instructions.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Derivative Assay, Western Blot, Lysis, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *

doi: 10.1074/jbc.M113.494542

Figure Lengend Snippet: Antibody-induced internalization and lysosomal degradation of Siglec-15 in osteoclasts. A, internalization of biotinylated Siglec-15. RAW264.7-derived osteoclast cell-surface proteins were labeled with a disulfide-linked biotinylation reagent. Cells were then treated with a combination of primary (anti-Siglec-15 B02 or control IgG) antibodies (at 4 °C) followed by secondary cross-linking antibodies (10 min at 37 °C, lanes 5 and 6), or with primary antibody alone (10 min at 37 °C, lanes 7 and 8). Control cells were incubated with B02, control IgG, or no antibody at 4 °C (lanes 1–3) without a 37 °C chase. After these antibody treatments, remaining cell-surface biotin was stripped with a reducing agent; for one sample, as an additional control (lane 4), this stripping step was omitted. Internalized, biotinylated proteins were collected from cell lysates by streptavidin immunoprecipitation, and Siglec-15 was detected by Western blotting. B, characterization of Siglec-15 endocytosis by confocal microscopy. RAW264.7-derived osteoclasts were cold-loaded with Siglec-15 antibody and either fixed immediately (No chase) or incubated in fresh, warm media for 10 or 45 min prior to fixation. Cells were then permeablized and stained with anti-LAMP2 and anti-human IgG (to detect internalized Siglec-15). C, Siglec-15 protein levels decrease rapidly following antibody treatment. Differentiated RAW264.7-derived osteoclasts were treated for the indicated times with anti-Siglec-15 or a control human IgG. Protein lysates were analyzed by Western blotting with the indicated antibodies. D, RAW264.7 differentiated in the presence of Siglec-15 antibodies show decreased Siglec-15 protein levels. Protein lysates of RAW264.7 cells, either non-differentiated (− RANKL) or differentiated (+ RANKL) in media with or without anti-Siglec-15 were analyzed by Western blotting with the indicated antibodies.

Article Snippet: Fab Preparation and Analysis Fab fragments were obtained from antibody B02 by ficin digest using the Mouse IgG1 Fab and F(ab′) 2 Preparation Kit (Pierce), according the manufacturer's instructions.

Techniques: Derivative Assay, Labeling, Incubation, Stripping Membranes, Immunoprecipitation, Western Blot, Confocal Microscopy, Staining

Journal: The Journal of Biological Chemistry

Article Title: Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *

doi: 10.1074/jbc.M113.494542

Figure Lengend Snippet: Siglec-15 antibodies inhibit osteoclast differentiation and activity in vitro. A, HOPs were grown with macrophage colony-stimulating factor alone (left panels) or with macrophage colony-stimulating factor and RANKL (to induce osteoclast differentiation) in the presence of control human IgG (middle panels) or anti-Siglec-15 (right panels) for 7 days. Cells were grown in parallel either on plastic (top panels) or on calcium phosphate-coated (bottom panels) plates. Multinucleated osteoclasts were identified on plastic by TRAP staining (red). Regions of calcium phosphate resorption were visualized by phase-contrast mode light microscopy after cell removal. B, upon prolonged treatment with RANKL (10 days rather than 7 days), HOPs grown in the presence of anti-Siglec-15 E09 fuse to form intensely TRAP-stained, multinucleated cells (right panel). The morphology of these cells is distinct from normal osteoclasts formed in the presence of a control antibody (middle panel). C, HOPs differentiated with RANKL in the presence of anti-Siglec-15 E09 display impaired TRAP secretion. TRAP levels in conditioned media of HOPs differentiated for 10 days were determined by ELISA. D, Siglec-15 antibody B02 inhibits differentiation of mouse RAW264.7 cells into osteoclasts. RAW264.7 cells were treated with RANKL to induce differentiation in the presence of control human IgG (middle panel) or anti-Siglec-15 B02 (right panel). Control cells were grown without RANKL or antibodies (left panel). After 3 days in culture, cells were stained for TRAP activity (red). The black arrows in the middle panel indicate examples of multinuclear, TRAP-positive osteoclasts.

Article Snippet: Fab Preparation and Analysis Fab fragments were obtained from antibody B02 by ficin digest using the Mouse IgG1 Fab and F(ab′) 2 Preparation Kit (Pierce), according the manufacturer's instructions.

Techniques: Activity Assay, In Vitro, Staining, Light Microscopy, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *

doi: 10.1074/jbc.M113.494542

Figure Lengend Snippet: Treatment with Siglec-15 antibodies increases bone mineral density in mice. Three- to 4-week-old male mice were treated with the B02 Siglec-15 antibody twice per week at 1, 3, or 10 mg/kg for 4 weeks. A, bone mineral density of long bones (left panel, right femur; middle panel, right tibia) and the lumbar vertebrae (L4–L6, right panel) were measured with a densitometer. The control group was treated similarly with a murine IgG at 10 mg/kg. B, cross-sectional images of a representative right femur (top panels) dissected from a control IgG-treated mouse (control IgG, left panel) or a mouse treated with 10 mg/kg of the B02 Siglec-15 antibody (Anti-Siglec-15, right panel). Similar cross-sectional images of the L5 vertebra from the same mice are shown in the lower panels. C, osteoclast-specific TRAP (TRAP 5b) activity was measured by ELISA in serum prepared from a terminal bleed. p values (versus the IgG treated group, n = 5/group) were calculated using Student's t test. *, p < 0.05; **, p < 0.02.

Article Snippet: Fab Preparation and Analysis Fab fragments were obtained from antibody B02 by ficin digest using the Mouse IgG1 Fab and F(ab′) 2 Preparation Kit (Pierce), according the manufacturer's instructions.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *

doi: 10.1074/jbc.M113.494542

Figure Lengend Snippet: Treatment with Siglec-15 antibody results in increased osteoclast number and osteoblast surface. A, representative photomicrographs (×10 magnification) of undecalcified mouse femur sections treated with control antibody (IgG) or anti-Siglec-15, histochemically stained with TRAP (A) and ALP (C). Scale bar, 100 μm. Histomorphometrical analysis was performed. B, N.Oc/BS, number of TRAP-positive osteoclasts per trabecular bone surface (mm). D, Ob.S/BS, ALP-positive osteoblast-surface per bone surface. p values (versus the IgG treated group, n = 5/group) were calculated using the Student's t test. *, p < 0.05.

Article Snippet: Fab Preparation and Analysis Fab fragments were obtained from antibody B02 by ficin digest using the Mouse IgG1 Fab and F(ab′) 2 Preparation Kit (Pierce), according the manufacturer's instructions.

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *

doi: 10.1074/jbc.M113.494542

Figure Lengend Snippet: Monovalent Fab fragments have reduced ability to induce degradation of Siglec-15 or inhibit osteoclast differentiation. A, flow cytometry analysis of IgG/Fab binding to Siglec-15 expressed on intact cells. Full-length Siglec-15 was expressed in 2936E cells by transient transfection and cells were labeled with the indicated concentrations of anti-Siglec-15 B02 full IgG or Fab fragments. The values in the graph correspond to the mean fluorescence intensity of the transfected cells. B, RAW264.7-derived osteoclasts were treated with the indicated concentrations of anti-Siglec-15 B02 IgG or Fab or a control human IgG (10 μg/ml) for 2 h. Protein lysates were analyzed by Western blotting with the indicated antibodies. C, RAW264.7 cells were differentiated in the presence of the indicated IgG and Fab concentrations and stained for TRAP expression.

Article Snippet: Fab Preparation and Analysis Fab fragments were obtained from antibody B02 by ficin digest using the Mouse IgG1 Fab and F(ab′) 2 Preparation Kit (Pierce), according the manufacturer's instructions.

Techniques: Flow Cytometry, Binding Assay, Transfection, Labeling, Fluorescence, Derivative Assay, Western Blot, Staining, Expressing