Journal: British Journal of Cancer

Article Title: Calcium-binding protein S100P is a new target gene of MACC1, drives colorectal cancer metastasis and serves as a prognostic biomarker

doi: 10.1038/s41416-022-01833-3

Figure Lengend Snippet: a – d Expression levels of MACC1 and S100P were determined by qRT-PCR and WB. Data show average gene expression ± SEM; N = 3. GAPDH served as a house-keeping gene, β-actin as loading control. Ectopic overexpression of MACC1 (left) in SW480 cells ( a ) lead to an upregulation S100P mRNA (centre) and protein levels (right), compared to control cells. Gene-specific knockdown of MACC1 (left) in SW620 cells ( b ) also decreased the S100P expression on mRNA (centre) and protein level (right). Ectopic overexpression of MACC1-GFP (left) in HT29 cells ( c ) resulted in increased mRNA (centre) and protein levels of S100P (right). Ectopic expression (left panel) or RNAi-based knockdown (right panel) in SW480 and SW620 cells ( d ), respectively, resulted in differential S100P expression. e – g Secreted S100P levels in the media of the respective cell lines were quantified with target-specific ELISA ( N = 4). Ectopic overexpression of MACC1, as well as S100P in SW480 cells ( e ) significantly increased the level of secreted S100P protein after 24-h incubation in serum-free medium, similarly to an ectopic expression of a MACC1-GFP fusion construct in HT29 cells ( f ). RNAi-mediated knockdown of MACC1, as well as S100P significantly reduced the amount of secreted S100P in the medium of the respective cells ( g ).

Article Snippet: The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) and incubated overnight with anti-human MACC1 (1:1000 dilution; Sigma-Aldrich) or S100P (1:250 dilution; R&D Systems, Minneapolis, MN), with β-actin (1:20,000 dilution; Pierce) as a loading control.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Enzyme-linked Immunosorbent Assay, Incubation, Construct

Journal: British Journal of Cancer

Article Title: Calcium-binding protein S100P is a new target gene of MACC1, drives colorectal cancer metastasis and serves as a prognostic biomarker

doi: 10.1038/s41416-022-01833-3

Figure Lengend Snippet: a Binding of MACC1 to the S100P promoter was determined by ChIP. A V5-specific antibody was Used to selectively pull-down ectopically overexpressed MACC1 in SW480/MACC1 cells (anti-V5). Pulling down V5 in SW480/vector cells or with unspecific human IgG in both cell lines (IgG) served as a negative control. Amplification of the S100P core promoter (−296 bp to +65 bp) indicates the binding of MACC1 to this fragment, while amplification of an unrelated GAPDH promoter sequence was used to test pull-down specificity. b MACC1- and S100P-related target genes were analysed in SW480/MACC1 and SW480/S100P cells by qRT-PCR and normalised to SW480/vector cells. Data show average values ± SEM; N = 3. c Elevated S100P expression increased the proliferation of SW480/S100P cells (upper panel), while knockdown of S100P decreased cell proliferation in SW620/shS100P cells (lower panel), compared to their respective control cells. Anchorage-dependent cell proliferation was determined by MTT assays. Data show average values ± SEM; N = 3. d Elevated S100P levels in SW480/S100P cells resulted in a higher ability of the cells to form colonies in soft agar (upper panel), while decreased S100P expression lead to fewer colonies of SW620/shS100P cells (lower panel), compared to their respective control cells. Single-cell suspensions were seeded in soft agar plates and colonies that were formed with more than four cells were counted. Representative images (×10 magnification) illustrate observed colony sizes. Data show average values ± SEM; N = 3. e S100P-overexpressing SW480/S100P cells showed higher cell migration through 12 μm pores of a transwell membrane, compared to SW480/vector cells (upper panel). SW620/shS100P cells with downregulated S100P expression migrated significantly less compared to control cells (lower panel). Cell migration was determined with Boyden chamber assays, counting the trans-migrated cells in the lower chamber. f SW480/S100P cells were able to close the wound within 4 days of incubation, compared to control cells (upper panel). The knockdown of S100P in SW620/shS100P cells resulted in decreased cell migration, in comparison to control cells (lower panel). In all, 300-µm wide wounds were inflicted in semi-confluent cell layers and wound closure was imaged daily. g SW480/S100P cells showed a higher ability to pass a matrigel-covered transwell membrane, compared to SW480/vector cells (upper panel). In turn, reduced S100P levels in SW620/shS100P resulted in decreased cell invasion, compared to control cells (lower panel). Data show average values ± SEM; N = 3.

Article Snippet: The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) and incubated overnight with anti-human MACC1 (1:1000 dilution; Sigma-Aldrich) or S100P (1:250 dilution; R&D Systems, Minneapolis, MN), with β-actin (1:20,000 dilution; Pierce) as a loading control.

Techniques: Binding Assay, Plasmid Preparation, Negative Control, Amplification, Sequencing, Quantitative RT-PCR, Expressing, Migration, Membrane, Incubation, Comparison

Journal: British Journal of Cancer

Article Title: Calcium-binding protein S100P is a new target gene of MACC1, drives colorectal cancer metastasis and serves as a prognostic biomarker

doi: 10.1038/s41416-022-01833-3

Figure Lengend Snippet: a SCID beige mice were intrasplenically transplanted with SW480/luc/vector upper panel) or SW480/luc/S100P cells (lower panel), with regular bioluminescence imaging to monitor tumour growth. b After sacrificing mice at day 49, spleens (tumour site) and livers (metastasis site) were removed and documented. Control mice ( N = 7; left panel) did not develop liver metastases, while all mice with transplanted SW480/luc/S100P cells ( N = 9; right panel) developed between one and six liver metastases. Ex vivo bioluminescence illustrated the tumour size and metastasis burden. c Quantification of tumour sizes showed an increased tumour growth (left panel), but also a significantly increased number of liver metastases (size <1 mm; right panel) for transplanted SW480/luc/S100P cells, compared to control cells. Data show average values ± SEM.

Article Snippet: The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) and incubated overnight with anti-human MACC1 (1:1000 dilution; Sigma-Aldrich) or S100P (1:250 dilution; R&D Systems, Minneapolis, MN), with β-actin (1:20,000 dilution; Pierce) as a loading control.

Techniques: Plasmid Preparation, Imaging, Ex Vivo

Journal: British Journal of Cancer

Article Title: Calcium-binding protein S100P is a new target gene of MACC1, drives colorectal cancer metastasis and serves as a prognostic biomarker

doi: 10.1038/s41416-022-01833-3

Figure Lengend Snippet: a CRC tissue sections of primary CRC tumours without (upper panel) or with (lower panel) metachronous metastasis were analysed for MACC1 and S100P expression by IHC. Representative tumour regions illustrate the correlation of MACC1 and S100P expression at ×20 and ×40 magnification. b S100P protein expression correlates with MACC1 expression in the described CRC patient cohort after IHC staining ( N = 27; solid line—linear regression; dotted line— 95% CI). c S100P protein expression is significantly increased in samples with high MACC1 expression. d S100P mRNA expression correlates with MACC1 expression in publicly available microarray datasets ( N = 308; solid line—linear regression; dotted line—95% CI).

Article Snippet: The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) and incubated overnight with anti-human MACC1 (1:1000 dilution; Sigma-Aldrich) or S100P (1:250 dilution; R&D Systems, Minneapolis, MN), with β-actin (1:20,000 dilution; Pierce) as a loading control.

Techniques: Expressing, Immunohistochemistry, Microarray

Journal: British Journal of Cancer

Article Title: Calcium-binding protein S100P is a new target gene of MACC1, drives colorectal cancer metastasis and serves as a prognostic biomarker

doi: 10.1038/s41416-022-01833-3

Figure Lengend Snippet: a S100P mRNA expression was found higher in tumours of patients (UICC I–III; N = 60) who developed metachronous metastases (left). Metastasis-free survival within 5 years after diagnosis was reduced when tumours express high S100P levels (centre). Combining MACC1 and S100P expression as prognostic biomarkers refines the identification of high-risk patients to develop metachronous metastasis (right). b Focusing on locally advanced tumours (UICC II–III; N = 43) the difference between tumours with and without metachronous metastasis became significant in S100P mRNA expression ( P < 0.05; left), which corresponds to significantly lower metastasis-free survival (centre) of patients with high intratumoural S100P expression. The combination of MACC1 and S100P mRNA expression as prognostic biomarkers refines the identification of high-risk patients to develop metachronous metastasis (right).

Article Snippet: The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) and incubated overnight with anti-human MACC1 (1:1000 dilution; Sigma-Aldrich) or S100P (1:250 dilution; R&D Systems, Minneapolis, MN), with β-actin (1:20,000 dilution; Pierce) as a loading control.

Techniques: Expressing

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: Reproductive Biology and Endocrinology : RB&E

Article Title: Down-regulation of S100P induces apoptosis in endometrial epithelial cell during GnRH antagonist protocol

doi: 10.1186/s12958-021-00787-0

Figure Lengend Snippet: S100P is down-regulated in the mid-secretory phase endometrium of participants from GnRH-ant group. A S100P gene expression in different groups from microarray data. B Immunohistochemical detection of S100P in the endometrium from the different groups, magnified in 200 times and the scale bar represents 50um. a. Negative control, b. Natural cycle control, c. GnRH antagonist group, d. GnRH agonist group; quantified in ( C ). D Location of S100P in endometrium by co-localization with vimentin and keratin. Red: S100P; Green: vimentin (b) and cytokeratin (f); Blue: nucleus. The scale bar represents 50um. E Western blot results of S100P and HOXA10 protein expression in endometrium from the natural cycle, GnRH-ant and GnRH-a groups. F Quantification of S100P protein expression. G Quantification of HOXA10 protein expression. * P < .05, ** P <.01, *** P <.001

Article Snippet: Briefly, the primary antibodies used are as follows: mouse anti-S100P monoclonal antibodies (MAB2957, R&D Systems, Minneapolis, MN, USA, 1:50 dilution), rabbit anti-cytokeratin monoclonal antibodies (ab181598, Abcam, UK, 1:100), rabbit anti-Vimentin monoclonal antibodies (ab92547, Abcam, UK, 1:100) and isotype antibody as negative control.

Techniques: Gene Expression, Microarray, Immunohistochemical staining, Negative Control, Control, Western Blot, Expressing

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Down-regulation of S100P induces apoptosis in endometrial epithelial cell during GnRH antagonist protocol

doi: 10.1186/s12958-021-00787-0

Figure Lengend Snippet: Knockdown of S100P induces apoptosis in endometrial epithelial cells. A Overexpression and knockdown of S100P mRNA was verified by Realtime PCR. B Overexpression and knockdown of S100P protein was verified and expression of Bcl-2 and Bax protein was analyzed by western blot. C Apoptosis rate was evaluated by flow cytometry after overexpression and knockdown of S100P in Ishikawa cells; results are quantified and analyzed in ( D ). NC: negative control using scramble sequence; KD: S100P knockdown; Mock: mock control transfected with empty plasmid vector; OE: S100P overexpression; * P < .05

Article Snippet: Briefly, the primary antibodies used are as follows: mouse anti-S100P monoclonal antibodies (MAB2957, R&D Systems, Minneapolis, MN, USA, 1:50 dilution), rabbit anti-cytokeratin monoclonal antibodies (ab181598, Abcam, UK, 1:100), rabbit anti-Vimentin monoclonal antibodies (ab92547, Abcam, UK, 1:100) and isotype antibody as negative control.

Techniques: Knockdown, Over Expression, Expressing, Western Blot, Flow Cytometry, Negative Control, Sequencing, Control, Transfection, Plasmid Preparation

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Down-regulation of S100P induces apoptosis in endometrial epithelial cell during GnRH antagonist protocol

doi: 10.1186/s12958-021-00787-0

Figure Lengend Snippet: GnRH antagonist-induced apoptosis in endometrial epithelial cells is rescued by S100P overexpression. A S100P concentration in the cell culture supernatant after GnRH-ant treatment, as determined by ELISA. B Protein expression of S100P, Bcl-2 and Bax in cultured cells after GnRH-ant treatment, as determined by western blot. C Flow cytometry analysis of apoptosis rate among different treatment groups; quantified and analyzed in ( D ). Mock: mock control transfected with empty plasmid vector; ANT: cells exposed to the GnRH antagonist- Cetrorelix; OE: S100P overexpression; * P < .05

Article Snippet: Briefly, the primary antibodies used are as follows: mouse anti-S100P monoclonal antibodies (MAB2957, R&D Systems, Minneapolis, MN, USA, 1:50 dilution), rabbit anti-cytokeratin monoclonal antibodies (ab181598, Abcam, UK, 1:100), rabbit anti-Vimentin monoclonal antibodies (ab92547, Abcam, UK, 1:100) and isotype antibody as negative control.

Techniques: Over Expression, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Flow Cytometry, Control, Transfection, Plasmid Preparation

Journal: BMC Cancer

Article Title: Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P

doi: 10.1186/s12885-020-07350-x

Figure Lengend Snippet: Sequences of primers used in the present study

Article Snippet: After permeabilization with 0.1% Triton X-100 for 45 min, cells were incubated with 1:50 mouse anti-human S100P monoclonal antibody (R&D Systems, Minneapolis, MN, USA) as the primary antibody, and then incubated with 1:500 Cy3-labeled goat anti-mouse IgG (Invitrogen) as the secondary antibody.

Techniques:

Journal: BMC Cancer

Article Title: Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P

doi: 10.1186/s12885-020-07350-x

Figure Lengend Snippet: Expression of S100P in endometrial cells. A: Immunofluorescence staining for S100P (red) and Hoechst33258 (blue) in EECs (a, b, c), ESCs (d, e, f), Ishikawa cells (g, h, i), and RL95–2 cells (j, k, l). PBS was used as a negative control (m, n, o), and the scale bar was 50 μm). B: The mRNA expression of S100P in different endometrial cells, as determined using qPCR. C: Fluorescence densitometric analysis using image J, normalized to that of EECs. EEC: primary endometrial epithelial cells, ESC: primary endometrial stromal cells, * P < 0.05

Article Snippet: After permeabilization with 0.1% Triton X-100 for 45 min, cells were incubated with 1:50 mouse anti-human S100P monoclonal antibody (R&D Systems, Minneapolis, MN, USA) as the primary antibody, and then incubated with 1:500 Cy3-labeled goat anti-mouse IgG (Invitrogen) as the secondary antibody.

Techniques: Expressing, Immunofluorescence, Staining, Negative Control, Fluorescence

Journal: BMC Cancer

Article Title: Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P

doi: 10.1186/s12885-020-07350-x

Figure Lengend Snippet: Expression of S100P in endometrial cancer and its precursor lesions. A: Immunohistochemistry demonstrating the distribution of S100P in endometrial cancer and its precursor lesions. a, Negative control; b, Normal endometrium in the proliferative phase; c, Endometrium of SH; d, Endometrium of CH; e, Endometrium of AH; f, Endometrium of EC (Magnified 200×). B: Densitometric analysis using image J. The data were normalized to the normal control. ns: no significance; * P < 0.05

Article Snippet: After permeabilization with 0.1% Triton X-100 for 45 min, cells were incubated with 1:50 mouse anti-human S100P monoclonal antibody (R&D Systems, Minneapolis, MN, USA) as the primary antibody, and then incubated with 1:500 Cy3-labeled goat anti-mouse IgG (Invitrogen) as the secondary antibody.

Techniques: Expressing, Immunohistochemistry, Negative Control, Control

Journal: BMC Cancer

Article Title: Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P

doi: 10.1186/s12885-020-07350-x

Figure Lengend Snippet: The efficiency of S100P knockdown and overexpression in cells. A, C: Knockdown of S100P (red) in Ishikawa or RL95–2 cells, respectively; Hoechst33258 (blue) for nuclear staining (Magnified 200 times). B, D: The changes in the S100P mRNA level in A or C, as determined using qPCR. ACTB (β-actin) was used as the internal reference (* P < 0.05). E, F: overexpression of S100P (red) in primary endometrial stromal cells and changes in the S100P mRNA level as mentioned above. Negative: uninfected cells; S100P shRNA: cells infected with S100P interference lentiviruses; S100P overexpression: cells infected with S100P overexpression lentiviruses; Control: Cells infected with scrambled sequence lentiviruses (A, B, C, D) or empty vector (E, F)

Article Snippet: After permeabilization with 0.1% Triton X-100 for 45 min, cells were incubated with 1:50 mouse anti-human S100P monoclonal antibody (R&D Systems, Minneapolis, MN, USA) as the primary antibody, and then incubated with 1:500 Cy3-labeled goat anti-mouse IgG (Invitrogen) as the secondary antibody.

Techniques: Knockdown, Over Expression, Staining, shRNA, Infection, Control, Sequencing, Plasmid Preparation

Journal: BMC Cancer

Article Title: Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P

doi: 10.1186/s12885-020-07350-x

Figure Lengend Snippet: Transwell experiments showing the effects of S100P on cell invasion. A: The invasive capacity of Ishikawa cells or endometrial stromal cells after silencing or overexpression of S100P using lentiviruses was determined using Transwell assays. Representative images (magnified 200 times) were captured at 48 h after the cells were seeded. All the experiments were performed in triplicate. a, b: ESCs transfected with the empty vector or showing S100P overexpression, respectively; c, d: Ishikawa cells with the scramble vector or subjected to S100P downregulation, respectively. B and C: Histogram showing the number of invasive cells per field from three independent experiments. * P < 0.05

Article Snippet: After permeabilization with 0.1% Triton X-100 for 45 min, cells were incubated with 1:50 mouse anti-human S100P monoclonal antibody (R&D Systems, Minneapolis, MN, USA) as the primary antibody, and then incubated with 1:500 Cy3-labeled goat anti-mouse IgG (Invitrogen) as the secondary antibody.

Techniques: Over Expression, Transfection, Plasmid Preparation

Journal: BMC Cancer

Article Title: Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P

doi: 10.1186/s12885-020-07350-x

Figure Lengend Snippet: The colocalization of S100P and Ezrin, and F-actin rearrangement after knocking down S100P . A: Laser confocal microscopy showing the colocalization (yellow) of S100P (green) and Ezrin (red) in RL95–2 cells. The panels below (e, f, g, h) are an enlargement of the box area (a, b, c, d). B and C: FITC-phalloidin (green) staining showing the rearrangement of F-actin in RL95–2 cells (left) and Ishikawa cells (right). Scramble control (a, c, e) or S100P shRNA (b, d, f) set as mentioned previously. The nuclei were counterstained using Hoechst33258 (blue). The magnification of (A) is displayed using a ruler and the magnification in B and C was 400 ×

Article Snippet: After permeabilization with 0.1% Triton X-100 for 45 min, cells were incubated with 1:50 mouse anti-human S100P monoclonal antibody (R&D Systems, Minneapolis, MN, USA) as the primary antibody, and then incubated with 1:500 Cy3-labeled goat anti-mouse IgG (Invitrogen) as the secondary antibody.

Techniques: Confocal Microscopy, Staining, Control, shRNA

Journal: BMC Cancer

Article Title: Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P

doi: 10.1186/s12885-020-07350-x

Figure Lengend Snippet: Sequences of primers used in the present study

Article Snippet: After permeabilization with 0.1% Triton X-100 for 45 min, rabbit anti-human Ezrin 1: 50 dilution (Epitomics, Burlingame, CA, USA; EP886Y) and mouse anti-human S100P 1:50 dilution (R&D Systems) were used as the primary antibodies.

Techniques:

Journal: BMC Cancer

Article Title: Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P

doi: 10.1186/s12885-020-07350-x

Figure Lengend Snippet: Expression of S100P in endometrial cells. A: Immunofluorescence staining for S100P (red) and Hoechst33258 (blue) in EECs (a, b, c), ESCs (d, e, f), Ishikawa cells (g, h, i), and RL95–2 cells (j, k, l). PBS was used as a negative control (m, n, o), and the scale bar was 50 μm). B: The mRNA expression of S100P in different endometrial cells, as determined using qPCR. C: Fluorescence densitometric analysis using image J, normalized to that of EECs. EEC: primary endometrial epithelial cells, ESC: primary endometrial stromal cells, * P < 0.05

Article Snippet: After permeabilization with 0.1% Triton X-100 for 45 min, rabbit anti-human Ezrin 1: 50 dilution (Epitomics, Burlingame, CA, USA; EP886Y) and mouse anti-human S100P 1:50 dilution (R&D Systems) were used as the primary antibodies.

Techniques: Expressing, Immunofluorescence, Staining, Negative Control, Fluorescence

Journal: BMC Cancer

Article Title: Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P

doi: 10.1186/s12885-020-07350-x

Figure Lengend Snippet: Expression of S100P in endometrial cancer and its precursor lesions. A: Immunohistochemistry demonstrating the distribution of S100P in endometrial cancer and its precursor lesions. a, Negative control; b, Normal endometrium in the proliferative phase; c, Endometrium of SH; d, Endometrium of CH; e, Endometrium of AH; f, Endometrium of EC (Magnified 200×). B: Densitometric analysis using image J. The data were normalized to the normal control. ns: no significance; * P < 0.05

Article Snippet: After permeabilization with 0.1% Triton X-100 for 45 min, rabbit anti-human Ezrin 1: 50 dilution (Epitomics, Burlingame, CA, USA; EP886Y) and mouse anti-human S100P 1:50 dilution (R&D Systems) were used as the primary antibodies.

Techniques: Expressing, Immunohistochemistry, Negative Control, Control

Journal: BMC Cancer

Article Title: Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P

doi: 10.1186/s12885-020-07350-x

Figure Lengend Snippet: The efficiency of S100P knockdown and overexpression in cells. A, C: Knockdown of S100P (red) in Ishikawa or RL95–2 cells, respectively; Hoechst33258 (blue) for nuclear staining (Magnified 200 times). B, D: The changes in the S100P mRNA level in A or C, as determined using qPCR. ACTB (β-actin) was used as the internal reference (* P < 0.05). E, F: overexpression of S100P (red) in primary endometrial stromal cells and changes in the S100P mRNA level as mentioned above. Negative: uninfected cells; S100P shRNA: cells infected with S100P interference lentiviruses; S100P overexpression: cells infected with S100P overexpression lentiviruses; Control: Cells infected with scrambled sequence lentiviruses (A, B, C, D) or empty vector (E, F)

Article Snippet: After permeabilization with 0.1% Triton X-100 for 45 min, rabbit anti-human Ezrin 1: 50 dilution (Epitomics, Burlingame, CA, USA; EP886Y) and mouse anti-human S100P 1:50 dilution (R&D Systems) were used as the primary antibodies.

Techniques: Knockdown, Over Expression, Staining, shRNA, Infection, Control, Sequencing, Plasmid Preparation

Journal: BMC Cancer

Article Title: Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P

doi: 10.1186/s12885-020-07350-x

Figure Lengend Snippet: Transwell experiments showing the effects of S100P on cell invasion. A: The invasive capacity of Ishikawa cells or endometrial stromal cells after silencing or overexpression of S100P using lentiviruses was determined using Transwell assays. Representative images (magnified 200 times) were captured at 48 h after the cells were seeded. All the experiments were performed in triplicate. a, b: ESCs transfected with the empty vector or showing S100P overexpression, respectively; c, d: Ishikawa cells with the scramble vector or subjected to S100P downregulation, respectively. B and C: Histogram showing the number of invasive cells per field from three independent experiments. * P < 0.05

Article Snippet: After permeabilization with 0.1% Triton X-100 for 45 min, rabbit anti-human Ezrin 1: 50 dilution (Epitomics, Burlingame, CA, USA; EP886Y) and mouse anti-human S100P 1:50 dilution (R&D Systems) were used as the primary antibodies.

Techniques: Over Expression, Transfection, Plasmid Preparation

Journal: BMC Cancer

Article Title: Promotion of the occurrence of endometrioid carcinoma by S100 calcium binding protein P

doi: 10.1186/s12885-020-07350-x

Figure Lengend Snippet: The colocalization of S100P and Ezrin, and F-actin rearrangement after knocking down S100P . A: Laser confocal microscopy showing the colocalization (yellow) of S100P (green) and Ezrin (red) in RL95–2 cells. The panels below (e, f, g, h) are an enlargement of the box area (a, b, c, d). B and C: FITC-phalloidin (green) staining showing the rearrangement of F-actin in RL95–2 cells (left) and Ishikawa cells (right). Scramble control (a, c, e) or S100P shRNA (b, d, f) set as mentioned previously. The nuclei were counterstained using Hoechst33258 (blue). The magnification of (A) is displayed using a ruler and the magnification in B and C was 400 ×

Article Snippet: After permeabilization with 0.1% Triton X-100 for 45 min, rabbit anti-human Ezrin 1: 50 dilution (Epitomics, Burlingame, CA, USA; EP886Y) and mouse anti-human S100P 1:50 dilution (R&D Systems) were used as the primary antibodies.

Techniques: Confocal Microscopy, Staining, Control, shRNA

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