goat anti s100p polyclonal antibody  (R&D Systems)

Journal: Biomolecules

Article Title: The Role of the C-Terminal Lysine of S100P in S100P-Induced Cell Migration and Metastasis

doi: 10.3390/biom11101471

Figure Lengend Snippet: Incidences of tumours and metastases.

Article Snippet: In some migration experiments, a polyclonal goat S100P antibody (Cat No. AF2957, R&D Systems, Abingdon, UK) was added to the culture medium at the concentration indicated in the Figure legends.

Techniques: Plasmid Preparation

Journal: Biomolecules

Article Title: The Role of the C-Terminal Lysine of S100P in S100P-Induced Cell Migration and Metastasis

doi: 10.3390/biom11101471

Figure Lengend Snippet: Immunofluorescence localisation of NMMIIA and S100P in cloned transfected cells. Rama 37 cells transfected with empty expression vector (Rama 37 + vector control; ( a – c )) or Rama 37 cells overexpressing wild-type S100P (Rama 37 + S100P; ( d – f )), K95A mutant S100P (Rama 37 + S100P K95A; ( g – i )), or ΔK95 mutant S100P (Rama 37 + S100P ΔK95; ( j – l )) were grown on fibronectin-coated coverslips for 48 h prior to fixation, permeabilisation, and staining using secondary antibody coupled to fluorescein isothiocyanate for NMMIIA ( a , d , g , j ) or Cy-3 for S100P ( b , e , h , k ). Cells were then mounted and viewed using a Zeiss LSM510 confocal laser scanning microscope. Merged images ( c , f , i , l ) are shown with overlaps in yellow. Arrows in ( a , c , g , j ) show filamental structures along the edge of the cell. The arrow in ( d ) shows foci of NMMIIA staining near the leading edge of the cell, which did not co-localise with S100P ( f ). The arrows in ( e , f ) show a large focus of S100P co-staining with NMMIIA in the perinuclear region. Bars, 10 μm in all panels.

Article Snippet: In some migration experiments, a polyclonal goat S100P antibody (Cat No. AF2957, R&D Systems, Abingdon, UK) was added to the culture medium at the concentration indicated in the Figure legends.

Techniques: Immunofluorescence, Clone Assay, Transfection, Expressing, Plasmid Preparation, Control, Mutagenesis, Staining, Laser-Scanning Microscopy

Journal: Biomolecules

Article Title: The Role of the C-Terminal Lysine of S100P in S100P-Induced Cell Migration and Metastasis

doi: 10.3390/biom11101471

Figure Lengend Snippet: Quantitation of focal adhesions in cell lines with and without metastatic potential.

Article Snippet: In some migration experiments, a polyclonal goat S100P antibody (Cat No. AF2957, R&D Systems, Abingdon, UK) was added to the culture medium at the concentration indicated in the Figure legends.

Techniques: Quantitation Assay, Plasmid Preparation, Control

Journal: Biomolecules

Article Title: The Role of the C-Terminal Lysine of S100P in S100P-Induced Cell Migration and Metastasis

doi: 10.3390/biom11101471

Figure Lengend Snippet: Effect of 6-aminocaproic acid, S100P antibody, aprotinin, or α-2-antiplasmin on migration of cells expressing wild-type and mutant S100P proteins. Transwell migration assays ( a , c ) or scratch migration assays in a Cell-IQ incubator ( b , d , e ) were carried out as described in Materials and Methods for cells expressing wild-type S100P (Rama 37 + S100P), K95A-mutant of S100P (Rama 37 + S100P K95A), ΔK95 mutant of S100P (Rama 37 + S100P ΔK95), or cells not expressing S100P (Rama 37 + vector control). In ( a , c ), the number of cells passing through to the underside of the membrane in 16 h were counted and are plotted as percentages of the mean number of untreated Rama 37 + vector control cells passing through the membrane. In ( b , d , e ), the times to scratch-wound closure are expressed as a percentage of the mean value for untreated Rama 37 + vector control cells. The effect on migration of cells of addition to the extracellular medium (upper and lower chambers in the case of Transwell assays) of 10 mM 6-aminocaproic acid (ACA, panels a , b ) or of an R&D Systems polyclonal goat S100P-specific antibody (Cat No. AF2957) at a concentration of 0.2 ng/μL in the medium (Antibody, panels a,b ) are shown. Each box and whisker plot shows the dispersion of data from 14 ( a ) or 11–12 ( b ) wells in two independent experiments carried out at different times. ( c ) Effect on Transwell migration of 50 μg/mL aprotinin (Aprotinin) or 10 μg/mL α-2-antiplasmin (Antiplasmin) in the extracellular medium. ( d ) Effect on scratch-wound migration of 25 μg/mL aprotinin (Aprotinin 25) or 50 μg/mL aprotinin (Aprotinin 50) in the extracellular medium. ( e ) Effect of 10 μg/mL α-2-antiplasmin (Antiplasmin) on scratch-wound migration. Each box and whisker plot shows the dispersion of data from 14 ( c ) or 14–25 wells ( d , e ) in two independent experiments carried out at different times. In all panels, p -values are indicated for comparison between the box and whisker plots beneath the ends of the horizontal lines as not significant (n.s., p > 0.05), or significant * ( p between 0.001 and 0.05) or ** ( p < 0.0001). For clarity, some p -value comparisons are shown without lines and in red colour, as follows: ( a ), n.s., Transwell assay Rama 37 + S100P K95A or Rama 37 +ΔK95 in the presence of 6-aminocaproic acid not significantly different from vector control (Dunnett post-hoc multiple comparison with a control); ** Rama 37 +S100P antibody significantly faster than Rama 37 + vector control, p < 0.0001; ( b ), scratch-wound assay, n.s. Rama 37 + S100P K95A or Rama 37 + ΔK95 in the presence of 6-aminocaproic acid not significantly different from vector control; ** Rama 37 +S100P antibody significantly faster than Rama 37 + vector control ( p < 0.0001). In all boxes, the black diamond and line show the median value; the cross shows the mean value. The white circles outside the whiskers denote outliers of >1.5 times the interquartile range.

Article Snippet: In some migration experiments, a polyclonal goat S100P antibody (Cat No. AF2957, R&D Systems, Abingdon, UK) was added to the culture medium at the concentration indicated in the Figure legends.

Techniques: Migration, Expressing, Mutagenesis, Plasmid Preparation, Control, Membrane, Concentration Assay, Whisker Assay, Dispersion, Comparison, Transwell Assay, Scratch Wound Assay Assay

Journal: Biomolecules

Article Title: The Role of the C-Terminal Lysine of S100P in S100P-Induced Cell Migration and Metastasis

doi: 10.3390/biom11101471

Figure Lengend Snippet: Association of S100P with cell membranes. ( A ) Total cell extracts and membrane fractions prepared as described in Materials and Methods from Rama 37 cells expressing S100P proteins and were subjected to Western blotting to detect S100P protein, the cytoplasmic marker, tubulin, and the plasma membrane marker, caveolin 1. ( B ) Living cells not expressing S100P (Rama 37 + vector control; panels a – c ) or expressing wild-type S100P (Rama 37 + S100P; panels d – f ), K95A mutant S100P (Rama 37 + S100P K95A; panels g – i ), or S100P protein with the C-terminal amino acid deleted (Rama 37 + S100P ΔK95; panels j – l ) were incubated with S100P antibody in the culture medium, and bound antibodies were detected with Cy-3-labelled secondary antibody, as described in Materials and Methods and observed with a Zeiss LSM510 confocal laser scanning microscope. Three separate typical fields are shown for each cell line. ( C ) Antibodies directed at S100P (panels a , f ) or cytoplasmic protein, eEF1A (panels c , h ), were added to the culture medium of living S100P-negative Rama 37 cells (Rama 37 + vector control; panels a , c ) or S100P-positive Rama 37 cells (Rama 37 + S100P; panels f , h ). Parallel cultures of S100P-negative cells (Rama 37 + vector control; panels b , d , e ) and S100P-positive cells (Rama 37 + S100P; panels g , i , j ) were permeabilised before being treated with the antibodies directed at S100P (permeabilised; panels b , g ) or eEF1A (permeabilised; panels d , e , i , j ). Following fixation, bound antibodies were detected with fluorescein isothiocyanate-conjugated secondary antibodies (Materials and Methods) and observed with a Zeiss LSM510 confocal laser scanning microscope. Panels ( e , j ) show the same fields as panels ( d , i ), but the intensity of the fluorescence signal has been reduced to show individual cells. Bars, ( B , C ) = 50 μm.

Article Snippet: In some migration experiments, a polyclonal goat S100P antibody (Cat No. AF2957, R&D Systems, Abingdon, UK) was added to the culture medium at the concentration indicated in the Figure legends.

Techniques: Membrane, Expressing, Western Blot, Marker, Clinical Proteomics, Plasmid Preparation, Control, Mutagenesis, Incubation, Laser-Scanning Microscopy, Fluorescence

Journal: Biomolecules

Article Title: The Role of the C-Terminal Lysine of S100P in S100P-Induced Cell Migration and Metastasis

doi: 10.3390/biom11101471

Figure Lengend Snippet: Effect of extracellular addition of S100P on migration of S100P non-expressing cells in Transwell and scratch-wound migration assays. ( a ) Transwell migration assays were carried out as described in Materials and Methods. The number of S100P-negative Rama 37 empty-vector-transfected control cells passing through to the underside of the membrane in 16 h in the presence of 10 mM 6-ACA (Rama 37 + vector + 10 mM 6-ACA), R&D Systems polyclonal goat S100P-specific antibody, Cat. No AF2957 at a concentration of 0.2 ng/μL in the medium (Rama 37 + vector + S100P antibody), 50 μg/mL aprotinin (Rama 37 + vector + 50 μg/mL aprotinin), or 20 μg/mL α-2-antiplasmin (Rama 37 + vector + 20 μg/mL antiplasmin), either in the absence (No S100P) or in the presence (+ S100P) of 10 μg/mL recombinant S100P (rS100P) were counted and are plotted as a percentage of the mean of untreated Rama 37 + vector control cells (Rama 37 + vector control). Each box and whisker plot shows the dispersion of data from 60 separate data points from 4 independent experiments (control, 6-ACA, S100P antibody) or 10 to 30 data points from 2 independent experiments (aprotinin and antiplasmin) carried out at different times. ( b ) Rama 37 cells transfected with empty expression vector were subjected to scratch-wound assays in the absence of or in the presence of various concentrations of rS100P added to the extracellular medium and the time-to-scratch-closure recorded for 9 separate wells in two independent experiments carried out at different times. For both ( a , b ), p -values are indicated for comparison between the box and whisker plots beneath the ends of the horizontal lines as n.s. not significant ( p > 0.05) or significant ** ( p < 0.0001). In ( a ), for clarity, some p -value comparisons are shown without lines and in red colour, as follows: n.s., no-S100P controls for 10 mM 6-ACA, 50 μg/mL aprotinin, and 20 μg/mL α-2-antiplasmin are not significantly different from the no-S100P, no-addition control ( p = 0.055, 0.946, 0.932, respectively, Dunnett post-hoc test), or * no S100P for 0.2 ng/μL S100P antibody, significantly faster than the no-S100P, no-addition control ( p = 0.0002). In all boxes for ( a , b ), the black diamond and line show the median value; the cross shows the mean value. The white circles outside the whiskers denote outliers of >1.5 times the interquartile range.

Article Snippet: In some migration experiments, a polyclonal goat S100P antibody (Cat No. AF2957, R&D Systems, Abingdon, UK) was added to the culture medium at the concentration indicated in the Figure legends.

Techniques: Migration, Expressing, Plasmid Preparation, Transfection, Control, Membrane, Concentration Assay, Recombinant, Whisker Assay, Dispersion, Comparison

Journal: Scientific Reports

Article Title: S100P enhances the motility and invasion of human trophoblast cell lines

doi: 10.1038/s41598-018-29852-2

Figure Lengend Snippet: S100P is expressed in trophoblasts including extravillous trophoblasts and more significantly during the first trimester in placental tissues. Expression of S100P proteins was analysed on both lysates ( A , B ) and sections ( C , E ) obtained from different paraffin-embedded placental samples from different gestational periods (first trimester (n = 5), second trimester (n = 4), or third trimester (n = 7). Proteins extracted from paraffin-embedded placental block sections (1 st trimester; 2 nd trimester and 3 rd trimester) at equal loading were separated by SDS-PAGE electrophoresis. Western blotting was carried out and membranes probed for S100P or α-tubulin and cropped images are presented ( A ). Levels of S100P were measured by densitometry analysis after Western blotting and normalised to α-tubulin for all samples (first trimester (n = 5), second trimester (n = 4), or third trimester (n = 7). Data is presented as percentage means ± SD of 2 independent experiments compared to the first trimester ( B ). Immunohistochemistry staining using a goat polyclonal S100P antibody and counterstaining on human placental tissues was performed as described in Methods. Arrows indicate cytotrophoblast cells (CT), syncytium (ST). Stroma is also highlighted. Bar corresponds to 150 μm ( C ). Quantification of S100P DAB staining and intensity in 1 st , 2 nd and 3 rd trimester serial sections. Data of an individual representative experiment is presented as the mean values ± SD of 3 independent samples ( D ). Statistical analysis ( B , D ) show ± SD compared to the first trimester samples of an individual representative experiment. *P < 0.05 and **P < 0.01 (one way- ANOVA). Immunohistochemistry staining using a panel of trophoblast marker antibodies (cytokeratin 7 (CK7), HLA-G, integrin α6 (CD49F) and S100P antibody) and counterstaining on anchoring columns of serial human placental tissues was performed as described in Methods. Arrows indicate cytotrophoblast cells (CT), syncytium (ST). Stroma is also highlighted. Bar corresponds to 100 μm ( E ).

Article Snippet: Non-specific protein binding was blocked by incubation with either 1% (v/v) normal bovine serum and 5% (v/v) goat IgG (Vector Labs, UK) or 10% (v/v) normal swine serum (Vector Labs, UK) for 1 hour and sections were then incubated with a goat polyclonal anti-S100P antibody (R&D, UK), a rabbit monoclonal anti-S100P antibody (Abcam, UK), a mouse monoclonal anti-HLA-G antibody (Abcam, UK), a rabbit polyclonal anti-CD49f (integrin α6) antibody (Abcam, UK) or an mouse monoclonal anti cytokeratin7 antibody (Leica Biosystems, UK) at 4 °C overnight (Supplementary Table ).

Techniques: Expressing, Blocking Assay, SDS Page, Electrophoresis, Western Blot, Immunohistochemistry, Staining, Marker

Journal: Scientific Reports

Article Title: S100P enhances the motility and invasion of human trophoblast cell lines

doi: 10.1038/s41598-018-29852-2

Figure Lengend Snippet: S100P is expressed in Jeg-3 and Bewo but not HTR8 EV trophoblast cell lines. HTR8, Bewo and Jeg-3 cells, along with HeLa A3 induced for S100P expression (or their non-expressing counterparts), were grown for 48 hours prior to collection for mRNA qPCR analysis ( A ) or 72 hours prior to collection for protein Western blotting ( B ). mRNAs were isolated using TRIS reagent followed by reverse transcription and quantitative PCR analysis using S100P and β-actin primers, as described in Methods. Data is presented as 2 ∆ CT mean values ± SD of 3 independent samples of a representative experiment compared to the non-induced HeLaA3 cells. **P < 0.0001 (one way- ANOVA). ( A ) For the protein levels, cells were collected and solubilised in Laemmeli buffer and equal loading were separated by SDS-PAGE electrophoresis. Western blotting was carried out and membranes probed for S100P or α-tubulin and cropped images are presented. ( B ) Expression levels of S100P were measured by densitometric analysis, normalised to α-tubulin and presented in comparison to the non-induced HeLa A3 cells as percentage mean values ± SD of 3 independent samples compared to the non-induced HeLa A3 cells. **P < 0.0001 (one way- ANOVA). ( C ) For immunostaining, Bewo and Jeg-3 cells, along with HeLa A3 induced for S100P expression (or their non-expressing counterparts) were seeded on fibronectin-coated coverslips and grown for 48 hours prior to fixation, permeabilisation and staining for S100P and actin. Cells were mounted and viewed using epifluorescence microscopy. ( D ) Images in the last row correspond to the focused regions of the highlighted cells. Bar corresponds to 25 μm.

Article Snippet: Non-specific protein binding was blocked by incubation with either 1% (v/v) normal bovine serum and 5% (v/v) goat IgG (Vector Labs, UK) or 10% (v/v) normal swine serum (Vector Labs, UK) for 1 hour and sections were then incubated with a goat polyclonal anti-S100P antibody (R&D, UK), a rabbit monoclonal anti-S100P antibody (Abcam, UK), a mouse monoclonal anti-HLA-G antibody (Abcam, UK), a rabbit polyclonal anti-CD49f (integrin α6) antibody (Abcam, UK) or an mouse monoclonal anti cytokeratin7 antibody (Leica Biosystems, UK) at 4 °C overnight (Supplementary Table ).

Techniques: Expressing, Western Blot, Isolation, Real-time Polymerase Chain Reaction, SDS Page, Electrophoresis, Immunostaining, Staining, Epifluorescence Microscopy

Journal: Scientific Reports

Article Title: S100P enhances the motility and invasion of human trophoblast cell lines

doi: 10.1038/s41598-018-29852-2

Figure Lengend Snippet: Specific knock-down of S100P in Jeg-3 and Bewo trophoblastic cell lines. Bewo and Jeg-3 cells were incubated in the presence of different S100P or control siRNAs for 48 hours prior to collection for mRNA qPCR analysis ( A , D ) or 72 hours prior to collection for protein Western blotting ( B , C , E , F ). mRNAs were isolated using TRIS reagent followed by reverse transcription and quantitative PCR analysis using primers for S100P and β-actin, as indicated in Methods. Data is presented as 2 ∆ CT mean values ± SD of 3 independent samples of a representative experiment compared to non-treated control samples. ***P < 0.0001 (one way- ANOVA) ( A , D ). For protein levels, cells were collected and solubilised in Laemmeli buffer and equal loading were separated by SDS-PAGE electrophoresis. Western blotting was carried out and membranes probed for S100P or α-tubulin and cropped images are presented ( B , E ). Expression levels of S100P were measured by densitometric analysis, normalised to α-tubulin and presented as percentage mean values ± SD of 3 independent samples of a representative experiment compared to non-treated control samples. *P < 0.01 ***P < 0.0001 (one way- ANOVA) ( C , F ).

Article Snippet: Non-specific protein binding was blocked by incubation with either 1% (v/v) normal bovine serum and 5% (v/v) goat IgG (Vector Labs, UK) or 10% (v/v) normal swine serum (Vector Labs, UK) for 1 hour and sections were then incubated with a goat polyclonal anti-S100P antibody (R&D, UK), a rabbit monoclonal anti-S100P antibody (Abcam, UK), a mouse monoclonal anti-HLA-G antibody (Abcam, UK), a rabbit polyclonal anti-CD49f (integrin α6) antibody (Abcam, UK) or an mouse monoclonal anti cytokeratin7 antibody (Leica Biosystems, UK) at 4 °C overnight (Supplementary Table ).

Techniques: Incubation, Western Blot, Isolation, Real-time Polymerase Chain Reaction, SDS Page, Electrophoresis, Expressing

Journal: Scientific Reports

Article Title: S100P enhances the motility and invasion of human trophoblast cell lines

doi: 10.1038/s41598-018-29852-2

Figure Lengend Snippet: Specific reduction of S100P by siRNA technology leads to significant impairment in cellular motility of Jeg-3 and Bewo trophoblast cells. Bewo and Jeg-3 cells were treated with different S100P siRNAs (siRNA4 and siRNA6) or mock-control for 48 hours prior to starvation with low serum-containing medium. 24 hours later, cells were seeded into Boyden chambers for 16 hours prior to fixation and staining using the Diffquik histochemical kit for labelling of both nuclei and cytoplasm. ( A , B ) 5 random fields were quantified for each chamber. Data is presented as means ± SEM of 4 independent experiments relative to controls (percentage) from 4 replicate wells for each set of conditions. ***P < 0.0001 compared to control and mock treated (one way-ANOVA). After siRNA delivery, and a further 48 hours incubation, cells were seeded on fibronectin-coated coverslips and grown for a further 48 hours prior to fixation, permeabilisation and staining for the focal adhesion marker paxillin and the cytoskeletal marker actin. Cells were mounted and viewed using epifluorescence microscopy. ( C ) Images on the last row correspond to the focused regions of the highlighted cells. Bar corresponds to 50 μm. Images of representative fields of motility/invasion assays were taken with the EVOS XL Cell Imaging System at x20 magnification.

Article Snippet: Non-specific protein binding was blocked by incubation with either 1% (v/v) normal bovine serum and 5% (v/v) goat IgG (Vector Labs, UK) or 10% (v/v) normal swine serum (Vector Labs, UK) for 1 hour and sections were then incubated with a goat polyclonal anti-S100P antibody (R&D, UK), a rabbit monoclonal anti-S100P antibody (Abcam, UK), a mouse monoclonal anti-HLA-G antibody (Abcam, UK), a rabbit polyclonal anti-CD49f (integrin α6) antibody (Abcam, UK) or an mouse monoclonal anti cytokeratin7 antibody (Leica Biosystems, UK) at 4 °C overnight (Supplementary Table ).

Techniques: Staining, Incubation, Marker, Epifluorescence Microscopy, Imaging

Journal: Scientific Reports

Article Title: S100P enhances the motility and invasion of human trophoblast cell lines

doi: 10.1038/s41598-018-29852-2

Figure Lengend Snippet: Specific reduction of S100P by siRNA leads to a reduction in distance of migration of Jeg-3 trophoblast cells. Jeg-3 cells treated with different S100P siRNAs (siRNA4 and siRNA6) or control mock for 48 hours were used either for quantitative chemotaxis-directed motility ( A , B ) or directional migration after wounding. ( C ) For chemotaxis, cells were first starved with low serum-containing medium for 24 hours prior to seeding on fibronectin coated coverslips in a Dunn chamber ( A , B ) against high serum containing medium. Digitised images were obtained using the Cell IQ automated image capture system, (Chip-Man Technologies) in which pre-selected fields were imaged using phase contrast microscopy on a continuous loop. Data collected was analysed using ImageJ and chemotaxis and migration software in order to analyse chemotaxis and distance of migration. Data is presented as mean values ± SD of 3 independent samples of a representative experiment compared to non-treated control samples. ***P < 0.0001 (one way-ANOVA). ( B ) For the wound healing experiment, cells were seeded on fibronectin-coated coverslips for 48 hours prior to attaining 100% confluency, scratched and grown for a further 16 hours prior to fixation, permeabilisation and staining for the focal adhesion marker paxillin and the cytoskeletal marker actin. Cells were mounted and viewed using epifluorescence microscopy. ( C ) Images in the last column correspond to the focused regions of the highlighted cells. Bar corresponds to 50 μm.

Article Snippet: Non-specific protein binding was blocked by incubation with either 1% (v/v) normal bovine serum and 5% (v/v) goat IgG (Vector Labs, UK) or 10% (v/v) normal swine serum (Vector Labs, UK) for 1 hour and sections were then incubated with a goat polyclonal anti-S100P antibody (R&D, UK), a rabbit monoclonal anti-S100P antibody (Abcam, UK), a mouse monoclonal anti-HLA-G antibody (Abcam, UK), a rabbit polyclonal anti-CD49f (integrin α6) antibody (Abcam, UK) or an mouse monoclonal anti cytokeratin7 antibody (Leica Biosystems, UK) at 4 °C overnight (Supplementary Table ).

Techniques: Migration, Chemotaxis Assay, Microscopy, Software, Staining, Marker, Epifluorescence Microscopy

Journal: Scientific Reports

Article Title: S100P enhances the motility and invasion of human trophoblast cell lines

doi: 10.1038/s41598-018-29852-2

Figure Lengend Snippet: Reduction of S100P in trophoblast cells leads to increases in the number of focal adhesions per cell.

Article Snippet: Non-specific protein binding was blocked by incubation with either 1% (v/v) normal bovine serum and 5% (v/v) goat IgG (Vector Labs, UK) or 10% (v/v) normal swine serum (Vector Labs, UK) for 1 hour and sections were then incubated with a goat polyclonal anti-S100P antibody (R&D, UK), a rabbit monoclonal anti-S100P antibody (Abcam, UK), a mouse monoclonal anti-HLA-G antibody (Abcam, UK), a rabbit polyclonal anti-CD49f (integrin α6) antibody (Abcam, UK) or an mouse monoclonal anti cytokeratin7 antibody (Leica Biosystems, UK) at 4 °C overnight (Supplementary Table ).

Techniques:

Journal: Scientific Reports

Article Title: S100P enhances the motility and invasion of human trophoblast cell lines

doi: 10.1038/s41598-018-29852-2

Figure Lengend Snippet: Reduction of S100P in trophoblast cells leads to increases in the number of focal adhesions per migrating cell.

Article Snippet: Non-specific protein binding was blocked by incubation with either 1% (v/v) normal bovine serum and 5% (v/v) goat IgG (Vector Labs, UK) or 10% (v/v) normal swine serum (Vector Labs, UK) for 1 hour and sections were then incubated with a goat polyclonal anti-S100P antibody (R&D, UK), a rabbit monoclonal anti-S100P antibody (Abcam, UK), a mouse monoclonal anti-HLA-G antibody (Abcam, UK), a rabbit polyclonal anti-CD49f (integrin α6) antibody (Abcam, UK) or an mouse monoclonal anti cytokeratin7 antibody (Leica Biosystems, UK) at 4 °C overnight (Supplementary Table ).

Techniques:

Journal: Scientific Reports

Article Title: S100P enhances the motility and invasion of human trophoblast cell lines

doi: 10.1038/s41598-018-29852-2

Figure Lengend Snippet: Modulation of S100P protein levels does not affect Jeg-3 and HTR8 trophoblast cell proliferation. Jeg-3 cells were treated with different S100P siRNAs (siRNA4 and siRNA6) or mock-control for 48 hours prior to seeding ( A ). Stably transfected HTR8/SVneo cells expressing S100P or the control counterpart were grown as described in methods ( B ). Cell lines were seeded into 24 well plates and left to grow for a further 24–48 hours before trypsinisation and removal from the wells and counting using trypan blue exclusion. Data is presented as percentage means ± SD of 3 independent experiments relative to controls from 3 replicate wells for each set of conditions. ***P < 0.0001 (one way-ANOVA).

Article Snippet: Non-specific protein binding was blocked by incubation with either 1% (v/v) normal bovine serum and 5% (v/v) goat IgG (Vector Labs, UK) or 10% (v/v) normal swine serum (Vector Labs, UK) for 1 hour and sections were then incubated with a goat polyclonal anti-S100P antibody (R&D, UK), a rabbit monoclonal anti-S100P antibody (Abcam, UK), a mouse monoclonal anti-HLA-G antibody (Abcam, UK), a rabbit polyclonal anti-CD49f (integrin α6) antibody (Abcam, UK) or an mouse monoclonal anti cytokeratin7 antibody (Leica Biosystems, UK) at 4 °C overnight (Supplementary Table ).

Techniques: Stable Transfection, Transfection, Expressing

Journal: Scientific Reports

Article Title: S100P enhances the motility and invasion of human trophoblast cell lines

doi: 10.1038/s41598-018-29852-2

Figure Lengend Snippet: Specific reduction of S100P by siRNA leads to significant impairment in invasive abilities of Jeg-3 and Bewo trophoblast cells. Jeg-3 ( A ) and Bewo ( B ) cells were treated with different S100P siRNAs (siRNA4 and siRNA6) or control siRNAs for 48 hours prior to starvation with low serum containing medium. 24 hours later, cells were seeded in Boyden chambers previously coated with matrigel and incubated for 16 hours prior to fixation and staining using the Diffquik histochemical kit for labelling of both nuclei and cytoplasm. ( A , B ) 5 random fields were quantified for each chamber. Data is presented as means ± SEM of 4 independent experiments relative to controls (percentage) from 4 replicate wells for each set of conditions. ***P < 0.0001 compared to control and mock treated (one way- ANOVA). Images of representative fields of motility/invasion assays were taken with the EVOS XL Cell Imaging System at x20 magnification.

Article Snippet: Non-specific protein binding was blocked by incubation with either 1% (v/v) normal bovine serum and 5% (v/v) goat IgG (Vector Labs, UK) or 10% (v/v) normal swine serum (Vector Labs, UK) for 1 hour and sections were then incubated with a goat polyclonal anti-S100P antibody (R&D, UK), a rabbit monoclonal anti-S100P antibody (Abcam, UK), a mouse monoclonal anti-HLA-G antibody (Abcam, UK), a rabbit polyclonal anti-CD49f (integrin α6) antibody (Abcam, UK) or an mouse monoclonal anti cytokeratin7 antibody (Leica Biosystems, UK) at 4 °C overnight (Supplementary Table ).

Techniques: Incubation, Staining, Imaging

Journal: Scientific Reports

Article Title: S100P enhances the motility and invasion of human trophoblast cell lines

doi: 10.1038/s41598-018-29852-2

Figure Lengend Snippet: Overexpression of S100P in HTR8/SVneo trophoblast cells leads to significant increases in motility and invasive abilities. Stable transfection of HTR8/SVneo cells with S100P cDNA in pcDNA3.1 hygromycin plasmid was established to isolate clones expressing different levels of S100P, or their counterpart control, and protein levels assessed by Western blotting ( A ). Cells were collected and solubilised in Laemmeli buffer at equal loading and were separated by SDS-PAGE electrophoresis. Western blotting was carried out and membranes probed for S100P or α-tubulin and cropped images are presented ( A ). Expression levels of S100P were measured by densitometric analysis, normalised to α-tubulin and presented in comparison to the control untreated equivalent cells ( B ). Error bars in ( B ) show ± standard deviation compared to untreated control samples from a representative experiment. ***P < 0.001 compared to control (one way- ANOVA). The same clones expressing different levels of S100P were seeded in either Boyden chambers alone or chambers previously coated with matrigel. Cells were incubated for 16 hours prior to fixation and staining using the Diffquik histochemical kit for labelling of both nuclei and cytoplasm. 5 random fields were quantified for each chamber. Data is presented as a percentage compared to the control untreated cells for cell motility ( C ) or invasion ( D ). Error bars in ( C , D ) show means ± SD of 3 independent experiments relative to controls (percentage) from 4 replicate wells for each set of conditions. **P < 0.005 and ***P < 0.0001 compared to control cells (one way- ANOVA). Images of representative fields of motility/invasion assays were taken with the EVOS XL Cell Imaging System at x20 magnification.

Article Snippet: Non-specific protein binding was blocked by incubation with either 1% (v/v) normal bovine serum and 5% (v/v) goat IgG (Vector Labs, UK) or 10% (v/v) normal swine serum (Vector Labs, UK) for 1 hour and sections were then incubated with a goat polyclonal anti-S100P antibody (R&D, UK), a rabbit monoclonal anti-S100P antibody (Abcam, UK), a mouse monoclonal anti-HLA-G antibody (Abcam, UK), a rabbit polyclonal anti-CD49f (integrin α6) antibody (Abcam, UK) or an mouse monoclonal anti cytokeratin7 antibody (Leica Biosystems, UK) at 4 °C overnight (Supplementary Table ).

Techniques: Over Expression, Stable Transfection, Plasmid Preparation, Clone Assay, Expressing, Western Blot, SDS Page, Electrophoresis, Standard Deviation, Incubation, Staining, Imaging