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: Statistical analysis was performed using GraphPad Prism 4.03 software.

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: Comparison of adhesion and migration in SW480 and SW620 genetically paired colon cancer cell lines. a Adhesion of SW480 and SW620 cells was assessed by absorbance at 590 nm (y-axis) of crystal violet stained adherent cells and a comparison between the adhesion capability of the cells after 8 h was compared. b A Comparison of migration capability of SW480 and SW620 cells measuring the cells occupying the wound area after 0, 12 and 24 represented as normalised particles per area over 24 h relative to particles at 0 h (calculated using ImageJ). Graphs are representative of three independent experiments carried out in triplicate showing consistent results. Error bars indicate the standard error in the mean A students t -test was performed using GraphPad Prism 4.03 software to assess statistical significance where n = 3. (** p < 0.01, *** p < 0.001)

Article Snippet: Statistical analysis was performed using GraphPad Prism 4.03 software.

Techniques: Migration, 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 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: Statistical analysis was performed using GraphPad Prism 4.03 software.

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: Statistical analysis was performed using GraphPad Prism 4.03 software.

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: Statistical analysis was performed using GraphPad Prism 4.03 software.

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