Journal: Oncology Letters

Article Title: Prognostic value of tumour endothelial markers in patients with endometrial cancer

doi: 10.3892/ol_00000037

Figure Lengend Snippet: Summary of tumour endothelial markers and primer characteristics.

Article Snippet: Primer and TEM data are summarised in . table ft1 table-wrap mode="anchored" t5 caption a7 Marker Name AB gene no. Amplicon size (bp) TEM-1 Endosialin, CD248 Hs00535574_s1 81 TEM-2 RASD family member 2 Hs00374755_m1 84 TEM-6 Tensin 3 Hs00224228_m1 60 TEM-7 Plexin domain containing 1 Hs00220723_m1 58 TEM-7r Plexin domain containing 2 Hs00262350_m1 109 TEM-8 Anthrax toxin receptor 1 Hs00216777_m1 67 GAPDH AP dehydrogenase Hs99999905_m1 122 β-actin β-actin Hs00242273_m1 93 L13A Ribosomal protein L13A Hs01926559_g1 105 PP14 Glycodelin Hs00171462_m1 109 MMP-2 Metalloproteinase 2 Hs00234422_m1 93 Open in a separate window Summary of tumour endothelial markers and primer characteristics.

Techniques: Amplification

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-C: Bacterially purified GST-Bub2 or GST-Bub2-Q132L, MBP-Bfa1 and 6xHis-Tem1 proteins were used to measure the kinetics of hydrolysis+dissociation (γ[ 32 P]GTP) or dissociation only (γ[ 35 S]GTP) using a filter binding assay (see ). Graphs show average values and standard deviations from three independent experiments. D: Exponentially growing cultures of the indicated strains were shifted to nocodazole containing medium at t = 0. Cell samples were withdrawn at the indicated time for FACS analysis of DNA contents. E: The percentage of cells with binucleate cell bodies accompanied or not by a checkpoint defect (indicated by re-budding in the absence of proper chromosome segregation) was scored in cycling cultures of the indicated strains shifted either to 14°C for 16h (left graph) or to 37°C for 3h (right graph). F-G: Exponentially growing cells with the indicated genotypes were arrested in G1 by α-factor and released into fresh medium at time 0. At 70’ after release α-factor was re-added to prevent cells from entering a second cell cycle. Cell samples were collected for FACS analysis of DNA contents (F) and for tubulin staining by indirect immunofluorescence (G). H: Cells were treated as in (F-G). TCA extracts were prepared from cell samples at the indicated time points to monitor kinetics of Bfa1-HA6 phosphorylation and Clb2 accumulation and degradation by western blot analysis. Pgk1 was used as loading control. I: Protein extracts from cells expressing the indicated tagged proteins were used for immunoprecipitation with an anti-HA affinity resin. Western blot analysis was then performed with anti-GFP and anti-HA antibodies. The input represents 1/25 th of the total extract used for each IP. J-K: Localization of eGFP- tagged Bub2/Bub2-Q132L, Tem1, Bfa1 (J) and Cdc15-GFP (K) was analysed by fluorescence microscopy after formaldehyde fixation.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Purification, Filter-binding Assay, Staining, Immunofluorescence, Western Blot, Expressing, Immunoprecipitation, Fluorescence, Microscopy

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-B: Cycling cells co-expressing Spc72-Bfa1-eGFPand Spc42-mCherry to mark the SPB (upper panel) or co-expressing Tem1-eGFP and Tub1-GFP (to mark microtubules, lower panel) were analysed to study the distribution of Spc72-Bfa1-eGFP (A) and Tem1-eGFP (B) at SPBs in SPC72-BFA1 bfa1Δ cells. C-D: Cycling cells with the indicated genotypes were shifted into nocodazole containing medium (t = 0). Cell samples were withdrawn at the indicated times for FACS analysis of DNA contents. E: The percentage of cells with binucleate cell bodies accompanied or not by a SPOC defect was scored after propidium iodide staining of cycling cultures of cells with the indicated genotypes after shift to 14°C for 16h. The histograms on the right side represent the DNA contents of the same cells as measured by FACS analysis. F: Percentage of metaphase cells with Cdc15-GFP at 0, 1 or 2 SPBs was scored in the indicated strains after formaldehyde fixation. Metaphases were identified by means of the Tub1-mCherry co-expressed marker. G: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YPD and incubated at the indicated temperature. H: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YP medium containing either glucose or galactose and incubated at 25°C for 48h.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Expressing, Staining, Marker, Incubation

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A: Bacterially purified 6XHis-Tem1 and 6XHis-Tem1-Q79L were loaded with γ[ 32 P]GTP either in the absence or in the presence of recombinant MBP-Bfa1 and incubated at 30°C for 10 minutes. The mixture was then added to GST-Bub2 or buffer alone and kinetics of GTP hydrolysis and dissociation was followed by a filter-binding assay (see details in ). Graphs show average values and standard deviations from three independent experiments. B: Wild type and TEM1-Q79L cells were arrested in G1 by α-factor and then released into fresh medium at 25°C (t = 0). Cell samples were withdrawn every 10’ to measure kinetics of budding and spindle formation/elongation after in situ immunostaining of tubulin. C: Actomyosin ring contraction has been visualized by live cell imaging of wild type and TEM1-Q79L expressing Myo1-GFP (n = 30). D: Logarithmically growing cultures of cells with the indicated genotypes were shifted into nocodazole containing medium (t = 0). DNA contents were analysed by flow cytometry at the indicated times. E: The percentage of cells with binucleate cell bodies accompanied or not by SPOC defect was scored after DAPI staining of cycling cells of the indicated strains shifted to 14°C for 16h. F: Logarithmically growing cultures of strains with the indicated genotypes were shifted to nocodazole containing medium (t = 0). DNA contents were analysed by flow cytometry at the indicated times. G: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YPD or YP galactose plates and incubated at 30°C for 48h.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Purification, Recombinant, Incubation, Filter-binding Assay, In Situ, Immunostaining, Live Cell Imaging, Expressing, Flow Cytometry, Staining

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: List of non-essential genes implicated in microtubules dynamics or spindle positioning identified in the SGA screen with TEM1-Q79L .

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Migration

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-B: Protein extracts from cells expressing the indicated tagged proteins were used for immunoprecipitation with an anti-HA affinity resin. Western blot analysis was then performed with anti-PK, anti-GFP and anti-HA antibodies. The input represents 1/25 th of the total extract used for each IP. C-F: Localization of eGFP- tagged Tem1 and Tem1-Q79L (C-D) or Bfa1-eGFP (E-F) was analysed in the indicated strains by fluorescence microscopy after formaldehyde fixation. Metaphase and anaphase cells were identified by means of the Tub1-mCherry co-expressed marker. Micrographs show representative cells of each strain in anaphase. G: Fluorescence intensity ratios were calculated between the two SPBs in anaphase cells of the indicated strains (see details in ).

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Expressing, Immunoprecipitation, Western Blot, Fluorescence, Microscopy, Marker

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-B: Distribution of Cdc15-GFP (A) or Mob1-GFP (B) was analysed in the indicated strains by fluorescence microscopy after formaldehyde fixation. Metaphase and anaphase cells were identified by means of the Tub1-mCherry co-expressed marker. Micrographs show representative wild type and TEM1-Q79L cells expressing Cdc15-GFP in metaphase. C: Serial dilutions of stationary phase cells with the indicated genotypes were spotted on YPD and incubated at the indicated temperatures for 48h.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Fluorescence, Microscopy, Marker, Expressing, Incubation

Journal: BMC Cancer

Article Title: TGFβ-mediated suppression of CD248 in non-cancer cells via canonical Smad-dependent signaling pathways is uncoupled in cancer cells

doi: 10.1186/1471-2407-14-113

Figure Lengend Snippet: Expression of CD248 by mesenchymal cells in response to cytokines and growth factors. Murine embryonic fibroblasts (MEF) (A) , 10 T1/2 cells (B) and murine aortic smooth muscle cells (SMC) (C) were incubated for 48 hrs with FGF (10 ng/ml), VEGF (20 ng/ml), PDGF (20 ng/ml), PMA (60 ng/ml), TGFβ (3 ng/ml), IL-6 (10 ng/ml), TNF-α (10 ng/ml), or IFN-γ (10 ng/ml). Cells were lysed and separated by SDS-PAGE under non-reducing conditions for Western immunoblotting to detect CD248 and phosphorylated Smad2. Equal loading was confirmed with actin control. Only TGFβ suppressed expression of CD248, while inducing phosphorylation of Smad2. Results are representative of 3 independent experiments. Molecular weight markers in kDa are shown on the left.

Article Snippet: Rabbit anti-human CD248 antibodies (Cat no #18160-1AP) were from ProteinTech (Chicago, USA); goat anti-human actin antibodies (#sc-1616) from Santa Cruz (USA); rabbit anti-SMAD1,5-Phospho (Cat no #9516), rabbit anti-Smad2-Phospho (#3101), rabbit anti-ERK1/2-phospho (#9101S), rabbit anti-p38-phospho (#9211), rabbit anti-SMAD2/3 (#5678) and rabbit anti-SMAD3 (#9513) were from Cell Signaling (USA).

Techniques: Expressing, Incubation, SDS Page, Western Blot, Control, Phospho-proteomics, Molecular Weight

Journal: BMC Cancer

Article Title: TGFβ-mediated suppression of CD248 in non-cancer cells via canonical Smad-dependent signaling pathways is uncoupled in cancer cells

doi: 10.1186/1471-2407-14-113

Figure Lengend Snippet: Expression of CD248 in response to increasing concentrations of TGFβ. (A) MEF were incubated for 48 hrs with increasing concentrations of TGFβ. Expression of CD248 (seen as monomers (~160 kDa) and dimers) and phosphorylation of Smad2, were detected by Western blot. (B) CD248 expression relative to actin expression was quantified by densitometry (n = 3 experiments) and results were normalized to the no-treatment condition. (C) CD248 expression by MEF (wild-type, WT; or lacking CD248, KO) was detected with specific anti-CD248 antibodies after exposure to carrier (Control) or TGFβ for 48 hrs. TGFβ suppresses CD248 in a concentration-dependent manner, with simultaneous increase in phosphorylated Smad2 and ERK1/2. Scale bar = 50 μm.

Article Snippet: Rabbit anti-human CD248 antibodies (Cat no #18160-1AP) were from ProteinTech (Chicago, USA); goat anti-human actin antibodies (#sc-1616) from Santa Cruz (USA); rabbit anti-SMAD1,5-Phospho (Cat no #9516), rabbit anti-Smad2-Phospho (#3101), rabbit anti-ERK1/2-phospho (#9101S), rabbit anti-p38-phospho (#9211), rabbit anti-SMAD2/3 (#5678) and rabbit anti-SMAD3 (#9513) were from Cell Signaling (USA).

Techniques: Expressing, Incubation, Phospho-proteomics, Western Blot, Control, Concentration Assay

Journal: BMC Cancer

Article Title: TGFβ-mediated suppression of CD248 in non-cancer cells via canonical Smad-dependent signaling pathways is uncoupled in cancer cells

doi: 10.1186/1471-2407-14-113

Figure Lengend Snippet: Temporal response of CD248 to TGFβ. (A) MEF were incubated for 0-48 hrs with TGFβ 3 ng/ml. Expression of CD248 and phosphorylation of Smad2, were detected by Western blot. (B) CD248 expression relative to actin expression was quantified by densitometry (n = 3 experiments) and results were normalized to the no-treatment condition. CD248 expression decreases as Smad2 is phosphorylated. (C) CD248 WT/WT (WT) or CD248 KO/KO (KO) MEF were exposed to TGFβ (0 or 3 ng/ml) for 48 hrs and lysates were Western blotted. Representative blots from 3 experiments are shown. Smad2 and ERK1/2 are phosphorylated in response to TGFβ even in cells that lack CD248.

Article Snippet: Rabbit anti-human CD248 antibodies (Cat no #18160-1AP) were from ProteinTech (Chicago, USA); goat anti-human actin antibodies (#sc-1616) from Santa Cruz (USA); rabbit anti-SMAD1,5-Phospho (Cat no #9516), rabbit anti-Smad2-Phospho (#3101), rabbit anti-ERK1/2-phospho (#9101S), rabbit anti-p38-phospho (#9211), rabbit anti-SMAD2/3 (#5678) and rabbit anti-SMAD3 (#9513) were from Cell Signaling (USA).

Techniques: Incubation, Expressing, Phospho-proteomics, Western Blot

Journal: BMC Cancer

Article Title: TGFβ-mediated suppression of CD248 in non-cancer cells via canonical Smad-dependent signaling pathways is uncoupled in cancer cells

doi: 10.1186/1471-2407-14-113

Figure Lengend Snippet: Stability of CD248 mRNA is unaffected by TGFβ. MEF were treated with TGFβ 3 ng/ml alone, α-amanatin 20 μg/ml alone, or with a combination of TGFβ and α-amanitin as described in Methods. CD248 mRNA levels, relative to the mRNA levels of the housekeeping gene GAPDH, were quantified at different time intervals by qRT-PCR. Results were normalized from 3 independent experiments, each done in triplicate. The half-life of CD248 mRNA is approximately 75 minutes, which is unaltered by TGFβ.

Article Snippet: Rabbit anti-human CD248 antibodies (Cat no #18160-1AP) were from ProteinTech (Chicago, USA); goat anti-human actin antibodies (#sc-1616) from Santa Cruz (USA); rabbit anti-SMAD1,5-Phospho (Cat no #9516), rabbit anti-Smad2-Phospho (#3101), rabbit anti-ERK1/2-phospho (#9101S), rabbit anti-p38-phospho (#9211), rabbit anti-SMAD2/3 (#5678) and rabbit anti-SMAD3 (#9513) were from Cell Signaling (USA).

Techniques: Quantitative RT-PCR

Journal: BMC Cancer

Article Title: TGFβ-mediated suppression of CD248 in non-cancer cells via canonical Smad-dependent signaling pathways is uncoupled in cancer cells

doi: 10.1186/1471-2407-14-113

Figure Lengend Snippet: TGFβ-induced suppression of CD248 is mediated via canonical signaling pathways. (A, B, C) MEF were incubated for 48 hrs with TGFβ 3 ng/ml and the ALK-inhibitor SB431542 1 μM either singly or in combination. Controls included carriers for SB431542 (DMSO) or for TGFβ (0.1% BSA). (A) Western blots and (B) immunofluorescence were used to detect expression of CD248 (green). (C) CD248 mRNA levels were also quantified (n = 3 experiments, each in triplicate; *p < 0.05). Results indicate that TGFβ-mediated suppression of CD248 protein and mRNA requires integrity of canonical ALK5-Smad2 signaling pathway. Scale bar = 50 μm.

Article Snippet: Rabbit anti-human CD248 antibodies (Cat no #18160-1AP) were from ProteinTech (Chicago, USA); goat anti-human actin antibodies (#sc-1616) from Santa Cruz (USA); rabbit anti-SMAD1,5-Phospho (Cat no #9516), rabbit anti-Smad2-Phospho (#3101), rabbit anti-ERK1/2-phospho (#9101S), rabbit anti-p38-phospho (#9211), rabbit anti-SMAD2/3 (#5678) and rabbit anti-SMAD3 (#9513) were from Cell Signaling (USA).

Techniques: Protein-Protein interactions, Incubation, Western Blot, Immunofluorescence, Expressing

Journal: BMC Cancer

Article Title: TGFβ-mediated suppression of CD248 in non-cancer cells via canonical Smad-dependent signaling pathways is uncoupled in cancer cells

doi: 10.1186/1471-2407-14-113

Figure Lengend Snippet: TGFβ-mediated suppression of CD248 via ALK5 is specific. (A, B) MEF were incubated with TGFβ (3 ng/ml) for 48 hrs in the presence or absence of the inhibitor of phosphorylated ERK1/2, U0126 10 μM (A) or phosphorylated p38, SB202190 10 μM (B) . Representative Western blots from 3 independent experiments are shown and were used to assess the effect on CD248 expression. TGFβ-coupling to either ERK1/2 or to p38 is not involved in its suppressive effects on CD248.

Article Snippet: Rabbit anti-human CD248 antibodies (Cat no #18160-1AP) were from ProteinTech (Chicago, USA); goat anti-human actin antibodies (#sc-1616) from Santa Cruz (USA); rabbit anti-SMAD1,5-Phospho (Cat no #9516), rabbit anti-Smad2-Phospho (#3101), rabbit anti-ERK1/2-phospho (#9101S), rabbit anti-p38-phospho (#9211), rabbit anti-SMAD2/3 (#5678) and rabbit anti-SMAD3 (#9513) were from Cell Signaling (USA).

Techniques: Incubation, Western Blot, Expressing

Journal: BMC Cancer

Article Title: TGFβ-mediated suppression of CD248 in non-cancer cells via canonical Smad-dependent signaling pathways is uncoupled in cancer cells

doi: 10.1186/1471-2407-14-113

Figure Lengend Snippet: Regulation of CD248 by BMP-2 and Activin. MEF were incubated with different concentrations of BMP2 (A) or activin (B) for 24 or 48 hrs. Representative Western blots from 3 independent experiments are shown and were used to assess the effect on CD248 expression.

Article Snippet: Rabbit anti-human CD248 antibodies (Cat no #18160-1AP) were from ProteinTech (Chicago, USA); goat anti-human actin antibodies (#sc-1616) from Santa Cruz (USA); rabbit anti-SMAD1,5-Phospho (Cat no #9516), rabbit anti-Smad2-Phospho (#3101), rabbit anti-ERK1/2-phospho (#9101S), rabbit anti-p38-phospho (#9211), rabbit anti-SMAD2/3 (#5678) and rabbit anti-SMAD3 (#9513) were from Cell Signaling (USA).

Techniques: Incubation, Western Blot, Expressing

Journal: BMC Cancer

Article Title: TGFβ-mediated suppression of CD248 in non-cancer cells via canonical Smad-dependent signaling pathways is uncoupled in cancer cells

doi: 10.1186/1471-2407-14-113

Figure Lengend Snippet: Regulation of CD248 in cancer cells. (A, B) Wehi-231 (A) and A20 (B) mouse lymphoma cells were incubated with different concentrations of TGFβ for 24 or 48 hrs and lysates were assessed by Western immunoblot. CD248 levels were minimally affected in spite of phosphorylation of Smad2. Results are representative of 3 independent experiments. (C) Normal fibroblasts (NF) and cancer associated fibroblasts (CAF) from murine mammary tissue were exposed to TGFβ for 24 or 48 hrs and CD248 mRNA levels were quantified and normalized to levels from untreated NF. CD248 mRNA levels in NF were significantly suppressed by TGFβ, whereas there was no effect on CD248 in CAF. *p < 0.05, n = 3.

Article Snippet: Rabbit anti-human CD248 antibodies (Cat no #18160-1AP) were from ProteinTech (Chicago, USA); goat anti-human actin antibodies (#sc-1616) from Santa Cruz (USA); rabbit anti-SMAD1,5-Phospho (Cat no #9516), rabbit anti-Smad2-Phospho (#3101), rabbit anti-ERK1/2-phospho (#9101S), rabbit anti-p38-phospho (#9211), rabbit anti-SMAD2/3 (#5678) and rabbit anti-SMAD3 (#9513) were from Cell Signaling (USA).

Techniques: Incubation, Western Blot, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Inflammatory Mesenchymal Stem Cells Express Abundant Membrane-Bound and Soluble Forms of C-Type Lectin-like CD248

doi: 10.3390/ijms24119546

Figure Lengend Snippet: Cell surface and intracellular distribution of CD248 in tumor cells and skin-derived MSCs (HFFFs). ( A ) RNA was extracted from a series of cell lines and CD248 expression examined by RT-PCR using CD248-specific primers. CD248-specific bands (711bp) were observed in the IMR-32 neuroblastoma and HFFF cell lines. Actin was used as a control reaction. ( B ) CD248 expression at the protein level was determined by flow cytometric staining of non-permeabilized cell lines with affinity-purified rabbit anti-CD248 followed by an RPE-conjugated secondary antibody. ( C ) CD248 localization within the cell was determined by double staining of permeabilized (acetone fixed) HFFFs on glass coverslips with affinity-purified rabbit anti-CD248 and a series of anti-organelle markers. TRITC-conjugated donkey anti-rabbit and FITC-conjugated donkey anti-mouse secondary antibodies were used. Expression was localized to the endosomal compartment by double staining with EEA1 and observing colocalization under a confocal microscope (white arrows). Magnification ×630.

Article Snippet: We performed double sequential IHC staining on the Leica Biosystems Bond-III automated staining system, using either the rabbit anti-Hu CD248 (clone E9Z7O, Ref 47948S Cell Signaling technology, Danvers, MA, USA), the rabbit anti-Hu ERG (clone EPR3864, Ventana, Cupertino, CA, USA), or the mouse antiCD45 (mouse monoclonal PA0042, Leica, Wetzlar, Germany).

Techniques: Derivative Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Staining, Affinity Purification, Double Staining, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Inflammatory Mesenchymal Stem Cells Express Abundant Membrane-Bound and Soluble Forms of C-Type Lectin-like CD248

doi: 10.3390/ijms24119546

Figure Lengend Snippet: CD248 expression by HFFFs in response to cytokines and growth factors: release of a cleaved soluble form. Quiescent HFFFs were treated for 24 h with 20 ng/mL rhVEGF 165 , 10 ng/mL bFGF, 1 ng/mL TGF-β1, 200 U/mL IL1-β, 50 ng/mL PMA, or were left untreated. ( A ) Flow cytometric staining of cells with affinity-purified rabbit anti-CD248, and expression of the results as fold changes in the mean of fluorescence over non-treated cells. ( B ) Western blot of whole cell lysates with affinity-purified rabbit anti-CD248 and comparison of the intensity of the CD248-specific band (160–170 kDa) following treatment with that of the non-treated cells showed no significant change in total cell CD248 expression following any treatment ( p > 0.05). Unequal loading was accounted for by blotting for α-tubulin (50 kDa band). The blots are representative of three separate experiments. The graph represents the mean change in intensity compared with non-treated cells ± SEM, n = 3. ( C ) TCS was collected from treated cells, freeze-dried and subjected to Western blotting with affinity-purified rabbit anti-CD248. Bands of 110 kDa were observed and the intensities of the bands following treatment were compared to the intensity of the band in non-treated cells. Histograms show the analysis of the band intensity of the WB ( n = 1). The blot and graph are representative of three separate experiments. The bars represent the fold increase in intensity over non-treated cells.

Article Snippet: We performed double sequential IHC staining on the Leica Biosystems Bond-III automated staining system, using either the rabbit anti-Hu CD248 (clone E9Z7O, Ref 47948S Cell Signaling technology, Danvers, MA, USA), the rabbit anti-Hu ERG (clone EPR3864, Ventana, Cupertino, CA, USA), or the mouse antiCD45 (mouse monoclonal PA0042, Leica, Wetzlar, Germany).

Techniques: Expressing, Staining, Affinity Purification, Fluorescence, Western Blot, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Inflammatory Mesenchymal Stem Cells Express Abundant Membrane-Bound and Soluble Forms of C-Type Lectin-like CD248

doi: 10.3390/ijms24119546

Figure Lengend Snippet: Matrix metalloproteases play a role in the generation of soluble CD248. HFFFs were treated for 4 h with 20 ng/mL rhVEGF 165 , 10 ng/mL bFGF, 1 ng/mL TGF-β1, 200 U/mL IL1-β, 50 ng/mL PMA, or were left untreated. ( A ) Cells were treated for 30 min with 1,10-phenanthroline prior to treatment together with growth factors and cytokines. TCS was collected, freeze-dried, and CD248 expression analyzed by Western blot probing with affinity-purified rabbit anti-CD248. Soluble CD248 is represented as a single band of 110 kDa. Histograms show the analysis of the band intensity of the above WB ( n = 1). ( B ) RNA was extracted from the cells and subjected to RT-PCR using primers specific for MMP-1, MMP-2, MMP-3, MMP-9, and TIMP-1 as well as actin primers as a control. PCR products were separated on an ethidium-bromide-stained 0.7% agarose gel and visualized under UV light.

Article Snippet: We performed double sequential IHC staining on the Leica Biosystems Bond-III automated staining system, using either the rabbit anti-Hu CD248 (clone E9Z7O, Ref 47948S Cell Signaling technology, Danvers, MA, USA), the rabbit anti-Hu ERG (clone EPR3864, Ventana, Cupertino, CA, USA), or the mouse antiCD45 (mouse monoclonal PA0042, Leica, Wetzlar, Germany).

Techniques: Expressing, Western Blot, Affinity Purification, Reverse Transcription Polymerase Chain Reaction, Control, Staining, Agarose Gel Electrophoresis

Journal: International Journal of Molecular Sciences

Article Title: Inflammatory Mesenchymal Stem Cells Express Abundant Membrane-Bound and Soluble Forms of C-Type Lectin-like CD248

doi: 10.3390/ijms24119546

Figure Lengend Snippet: CD248, CD90, and PDPN expression by synovial MSCs in response to inflammatory mediators. MSCs were stimulated with either IL1-β, TNF-α, TGFβ1, or IFN-γ (all at 20 ng/mL) or unstimulated during 6, 24, 48, or 72 h. Total RNA was extracted, and qRT-PCR was performed. Graphs represent the mean change in mRNA expression of ( A ) CD248, ( B ) CD90, and ( C ) PDPN compared to unstimulated MSCs ± SEM, n = 3. GAPH was used as the housekeeping gene. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: We performed double sequential IHC staining on the Leica Biosystems Bond-III automated staining system, using either the rabbit anti-Hu CD248 (clone E9Z7O, Ref 47948S Cell Signaling technology, Danvers, MA, USA), the rabbit anti-Hu ERG (clone EPR3864, Ventana, Cupertino, CA, USA), or the mouse antiCD45 (mouse monoclonal PA0042, Leica, Wetzlar, Germany).

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Inflammatory Mesenchymal Stem Cells Express Abundant Membrane-Bound and Soluble Forms of C-Type Lectin-like CD248

doi: 10.3390/ijms24119546

Figure Lengend Snippet: CD248 expression by synovial MSCs from arthritis patients. ( A ) OA and RA synovial MSCs/fibroblasts (RASF or OA SF) were grown on glass coverslips, acetone-fixed, and stained for CD248 using P1A6 (OA) or rabbit anti-CD248 (RA), followed by FITC-conjugated donkey anti-mouse or TRITC-conjugated donkey anti-rabbit respectively. Staining was visualized by confocal microscopy. Magnification ×400. Synovial fibroblasts were double-stained by flow cytometry with affinity-purified rabbit anti-CD248 and mouse anti-CD90 (Thy-1), followed by RPE-conjugated goat anti-rabbit and FITC-conjugated goat anti-mouse secondary antibodies. The staining was analyzed on a FACSCalibur instrument. The percentage of gated cells in each region is indicated. ( B ) soluble CD248 forms (110 kDa major band and two further cleaved products) were also detected in the supernatants of RA fibroblast cultures (passage 4) in response to IL1-β (24 h treatment). However, an MMP-3-specific inhibitor failed to control CD248 cleavage. ( C ) CD248 was immunoprecipitated from the synovial fluid using P7C5 bound to protein G Sepharose. The immunoprecipitated protein was detected by Western blot probing with affinity-purified rabbit anti-CD248, revealing a band of 110 kDa representing soluble CD248 (lane 1). A Western blot of lysates prepared from the synovial tissue of an OA and RA patient revealed a band of >120 kDa.

Article Snippet: We performed double sequential IHC staining on the Leica Biosystems Bond-III automated staining system, using either the rabbit anti-Hu CD248 (clone E9Z7O, Ref 47948S Cell Signaling technology, Danvers, MA, USA), the rabbit anti-Hu ERG (clone EPR3864, Ventana, Cupertino, CA, USA), or the mouse antiCD45 (mouse monoclonal PA0042, Leica, Wetzlar, Germany).

Techniques: Expressing, Staining, Confocal Microscopy, Flow Cytometry, Affinity Purification, Control, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Inflammatory Mesenchymal Stem Cells Express Abundant Membrane-Bound and Soluble Forms of C-Type Lectin-like CD248

doi: 10.3390/ijms24119546

Figure Lengend Snippet: CD248 is abundantly expressed by synovial MSCs in RA inflammatory tissues. ( a – o ): Frozen synovial tissue sections from an RA patient were stained with affinity-purified mouse (P1A6) or affinity-purified rabbit anti-CD248 (white arrows). Sections were double-stained using antibodies against CD90 (Thy-1), CD31, CD93, and VEGF followed by TRITC-conjugated and FITC-conjugated. Images were viewed using a fluorescent DMLB microscope. Insets in panels a and b depict background stainings using irrelevant antibodies. Magnification ×100 ( a – c ); ×200 ( d – f ), and ×400 for panels ( g – o ). ( p – r ): RA fibroblasts cultured on glass coverslips were double-stained for CD248 (affinity-purified rabbit antibody) and monoclonal antibodies against either CD141, CD63, or EEA1. Magnification (×400 for ( p , q ) and ×1000 for panel ( r )). Irrelevant antibodies failed to stain fibroblast cells (inset, panel ( p )).

Article Snippet: We performed double sequential IHC staining on the Leica Biosystems Bond-III automated staining system, using either the rabbit anti-Hu CD248 (clone E9Z7O, Ref 47948S Cell Signaling technology, Danvers, MA, USA), the rabbit anti-Hu ERG (clone EPR3864, Ventana, Cupertino, CA, USA), or the mouse antiCD45 (mouse monoclonal PA0042, Leica, Wetzlar, Germany).

Techniques: Staining, Affinity Purification, Microscopy, Cell Culture, Bioprocessing

Journal: International Journal of Molecular Sciences

Article Title: Inflammatory Mesenchymal Stem Cells Express Abundant Membrane-Bound and Soluble Forms of C-Type Lectin-like CD248

doi: 10.3390/ijms24119546

Figure Lengend Snippet: Paraffin wax RA tissue sections double-stained for CD248 and either CD45 (leukocytes) or endothelial (ERG) cell markers. Serial formalin-fixed paraffin-embedded (FFPE) sections were processed using sequential alkaline-phosphatase-based immunostaining with anti-CD248 (clone E9Z7O, red staining) and diaminobenzidine-based immunostaining with ( a ) anti-CD45 (clone X16/99, Leica) or ( b ) anti-ERG (clone EPR3864, Ventana).

Article Snippet: We performed double sequential IHC staining on the Leica Biosystems Bond-III automated staining system, using either the rabbit anti-Hu CD248 (clone E9Z7O, Ref 47948S Cell Signaling technology, Danvers, MA, USA), the rabbit anti-Hu ERG (clone EPR3864, Ventana, Cupertino, CA, USA), or the mouse antiCD45 (mouse monoclonal PA0042, Leica, Wetzlar, Germany).

Techniques: Paraffin Wax, Staining, Formalin-fixed Paraffin-Embedded, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Inflammatory Mesenchymal Stem Cells Express Abundant Membrane-Bound and Soluble Forms of C-Type Lectin-like CD248

doi: 10.3390/ijms24119546

Figure Lengend Snippet: Perivascular synovial MSCs (pMSCs) express CD248 (endosialin) and may remain at the perivascular level through the multimerin–CD93 anchoring complex. In inflammatory conditions (e.g., RA), interleukin-1 beta produced by macrophages and stimulating the release of MMP will contribute to the shedding of CD248. Membrane CD248 is known to control the PDGF-induced proliferation of MSCs (switching to an aggressive phenotype involved in pannus formation) but the role of sCD248 in this process or as a decoy receptor remains to be ascertained. Microscope image is shown at ×400.

Article Snippet: We performed double sequential IHC staining on the Leica Biosystems Bond-III automated staining system, using either the rabbit anti-Hu CD248 (clone E9Z7O, Ref 47948S Cell Signaling technology, Danvers, MA, USA), the rabbit anti-Hu ERG (clone EPR3864, Ventana, Cupertino, CA, USA), or the mouse antiCD45 (mouse monoclonal PA0042, Leica, Wetzlar, Germany).

Techniques: Produced, Membrane, Control, Microscopy