Journal: Nature Communications

Article Title: Hox11 expressing regional skeletal stem cells are progenitors for osteoblasts, chondrocytes and adipocytes throughout life

doi: 10.1038/s41467-019-11100-4

Figure Lengend Snippet: Hoxa11eGFP expression defines a continuous stromal population. a – f Hoxa11eGFP expression in the forelimb zeugopod (radius and ulna) shown from embryonic to adult stages with proximal on left and distal on right in all images. Hoxa11eGFP expression in radius and ulna a – c , higher magnification images show cartilage marker, Sox9 at E13.5 ( a , red) and osteoblast marker, Osterix at E14.5 ( b , magenta). d – f Mid-diaphysis radius (r) and ulna (u) (higher magnification images of mid-diaphysis ulna, white dashed box, shown on right). g Hoxa11eGFP and Osterix (red) at 8 weeks, white arrowheads identify individual Osterix positive nuclei. h Hoxa11eGFP (green) in E15.5 distal growth plate and higher magnification of perichondrium (white boxed region, bracket), 8 weeks periosteum (bracket), and P4 trabeculae. i , j Periosteal Hoxa11eGFP and Periostin (red) at 2 week ( i ) and 8 weeks ( j ). Dashed white lines mark periosteal boundary, dotted line separates inner and outer layers. j Cell marked by asterisk magnified in inset. In all images, green: Hoxa11eGFP, gray: DAPI. Bone marrow: bm, periosteum: po, endosteum: endo, cortical bone: cb, perichondrium: pc. Scale bars: ( a , b , d – f left panels, h growth plate), 200 μm, ( c ) 500 μm, ( d – f right panels, h trabecular bone) 100 μm, ( g , h periosteum, i , j ) 50 μm

Article Snippet: Sections were blocked with donkey serum and incubated with primary antibodies overnight at 4 °C against Sox9 (Millipore, AB5535, 1:500), Osterix (Abcam, ab22552, 1:300), Periostin (R&D Systems, AF2955, 1:400) and Perilipin (Sigma, P1873, 1:100).

Techniques: Expressing, Marker

Journal: Nature Communications

Article Title: Hox11 expressing regional skeletal stem cells are progenitors for osteoblasts, chondrocytes and adipocytes throughout life

doi: 10.1038/s41467-019-11100-4

Figure Lengend Snippet: Hoxa11 -lineage cells regenerate skeleton following fracture. Ulnar fracture was performed at 8 weeks of age ( a – f ) or 10 months ( g – k ) on Hoxa11-CreER T2 ;ROSA-tdTomato or Hoxa11eGFP;Hoxa11-CreER T2 ;ROSA-tdTomato mice following tamoxifen dosing at ( a – c ) E13.5—to pregnant female, or ( d – k ) P3. a , d , g Hoxa11 lineage-positive cells (Hoxa11iTom, red) within the fracture callus 10 days post-injury (DPI). Fracture line marked with dashed yellow line and cortical bone (cb), bone marrow (bm), and periosteum (po) are labeled. Dashed white lines indicate regions visualized with antibodies (green) for cartilage (Sox9, b , e , h ) and bone (Osx, c , f , i ). Higher magnification of regions marked by dashed white lines ( b , c , e , f ) with channels separated shown to right. ( j ) Expression of Hoxa11eGFP (green) and Hoxa11iTom within the expanded stromal population. White dashed box magnified in inset. k Expression of Periostin (green) and Hoxa11iTom within the expanded periosteal compartment (bracket). In all images, gray: DAPI. Scale bars: ( a , d , g ) 500 μm, ( b , c , e , f ) 200 μm, ( j ) 100 μm, ( h , i , k ) 50 μm

Article Snippet: Sections were blocked with donkey serum and incubated with primary antibodies overnight at 4 °C against Sox9 (Millipore, AB5535, 1:500), Osterix (Abcam, ab22552, 1:300), Periostin (R&D Systems, AF2955, 1:400) and Perilipin (Sigma, P1873, 1:100).

Techniques: Labeling, 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: Molecular Vision

Article Title: Extremely varied phenotypes in granular corneal dystrophy type 2 heterozygotes

doi:

Figure Lengend Snippet: Expression of transforming growth factor-beta-induced-gene protein (TGFBIp) in ten different normal corneal fibroblasts (NCF). A , B : western blots show evidence of differential expression of TGFBIp in cultured NCF-1 (37 year-old man), NCF-2 (62 year-old man), NCF-3 (20 year-old woman), NCF-4 (46 year-old man), NCF-5 (29 year-old man), NCF-6 (60 year-old man), NCF-7 (man with unknown-age), NCF-8 (woman with unknown-age), NCF-9 (69 year-old woman), NCF-10 (81 year-old man) from normal donor corneas. In the second experiment, NCF-4 was reloaded for a comparison of the two separate experiments. C , D : Relative density of TGFBIp expression of A and B , respectively. Relative density of NCF-4 was adjusted to 100% in both C and D for a comparison of the densities of normal corneal fibroblasts in two separate experiments. Blots containing 50 μg total protein from normal corneal fibroblasts were incubated with anti-TGFBIp antibody. TGFBIp expression was assayed using anti-TGFBIp polyclonal goat antibodies. β-actin has been used as a control for equal loading. Numbers on the right correspond to the molecular weight markers in kilodaltons (kDa).

Article Snippet: Equal amounts of proteins were then transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA), blocked in 5% dry milk in Tris-buffered saline containing Tween-20 (TBS-T) at room temperature for one hour, and then incubated with primary antibodies to TGFBIp (0.2 μg/ml; Cat. No. AF2935; R&D Systems, Minneapolis, MN) and β-actin (1:5,000 dilution; Cat. No. A-5441; Sigma, St Louis, MO) overnight at 4 °C.

Techniques: Expressing, Western Blot, Quantitative Proteomics, Cell Culture, Comparison, Incubation, Control, Molecular Weight