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: Molecular Neurodegeneration

Article Title: LRP1 is a neuronal receptor for α-synuclein uptake and spread

doi: 10.1186/s13024-022-00560-w

Figure Lengend Snippet: LRP1 regulates α-Syn uptake in iPSNs. a and b , α-Syn uptake in WT and LRP1 -KO iPSNs measured by flow cytometry (100 nM, 3 h of treatment). c Representative images of WT or LRP1 -KO iPSNs after α-Syn uptake. Scale bars, 20 μm. d and e , Transferrin (Tfn) uptake in WT and LRP1 -KO iPSNs measured by flow cytometry (300 nM, 3 h of treatment). f Uptake of α-Syn and Tfn in the presence of increasing concentrations of RAP. g EM images showing the structure of α-Syn oligomers and preformed fibrils (PFFs) used in panel h. Scale bars, 200 nm. h Uptake of α-Syn oligomers and PFFs in WT and LRP1 -KO iPSNs (100 nM monomer equivalent, 3 h of treatment). All experiments in ( a , b , d , e , f and h ) were performed in technical duplicates or triplicates over three independent experiments. All data are expressed as mean ± s.d. with individual data points shown. Data were analyzed by One-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The morphology of α-Syn species was confirmed by negative stain transmission electron microscopy. α-Syn oligomers (StressMarq, cat# SPR-484) and PFFs (StressMarq, cat# SPR-322-C) were prepared at 25 µM in water.

Techniques: Flow Cytometry

Journal: Translational Neurodegeneration

Article Title: Ultrasensitive detection of aggregated α-synuclein using quiescent seed amplification assay for the diagnosis of Parkinson’s disease

doi: 10.1186/s40035-024-00426-9

Figure Lengend Snippet: The detection limit of QSAA. a Schematic of the detection of quiescent amplification products by fluorescence. b , c Direct observation of amplification products under a fluorescence microscope. hPFFs/mPFFs (0.01 μg/ml) were incubated with 1 mg/ml human monomer (HM)/ mouse monomer (MM) and 40 μM ThT. QSAA was carried out at 70℃ for 24 h in the presence or absence of 10% AS. d , e TEM was performed to evaluate the fibrillar structure of the amplification products in ( b , c ). f QSAA using different seeds (100 fg αSyn hPFFs, 100 pg βSyn PFFs, 100 pg γSyn PFFs, 100 pg Aβ PFFs, 100 pg ζ306 (2R) PFFs, 100 pg K19CFh (3R) PFFs, 100 pg mixture of ζ306 PFFs and K19CFh PFFs). Data are presented as mean ± SD ( n = 4). g Normalized maximum ThT fluorescence values for QSAA in ( f ). h The lag phase data from ( g ) were used to calculate the PAR (1/h) for both SAA and QSAA reactions. For reactions that did not produce ThT fluorescence surpassing the threshold (assigned a lag phase of 36 hours), the rate was established at 0.027. i , k Serial dilutions of the hPFFs/mPFFs (equivalent to 1 ng, 10 pg, 1 pg, and 100 fg PFFs) were added to the quiescent incubation system, together with 1 mg/ml HM/MM, 10 mM Tris-HCl pH 7.5, 50 mM NaCl, and 40 μM ThT in a total volume of 20 μl. Fluorescence images were collected at 24 h of QSAA reaction. Panels below the fluorescence images are schematic diagrams illustrating the amplification products. j , l Calculation of the relative fluorescence area of filamentous or lamellar amplification products within circular droplets. Data presented as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test

Article Snippet: Human recombinant β-synuclein (βSyn) PFFs (Type 1) (Cat# SPR-457), human recombinant γ-synuclein (γSyn) PFFs (Type 1) (Cat# SPR-459), and human synthetic amyloid β 1-42 (Aβ 1-42 ) PFFs (Cat# SPR-487) were obtained from StressMarq Biosciences (British Columbia, Canada).

Techniques: Amplification, Fluorescence, Microscopy, Incubation, Comparison