Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CD44 acts as a coreceptor for cell-specific enhancement of signaling and regulatory T cell induction by TGM1, a parasite TGF-β mimic.

doi: 10.1073/pnas.2302370120

Figure Lengend Snippet: Fig. 2. Identification of CD44 as coreceptor for TGM1 signaling. (A) Ability of TGM1 (10 ng/mL) and TGF-β (1 ng/mL) to activate SMAD signaling in four cell lines, 771 (murine B cell lymphoma), EL4 (murine thymoma), NM18 (mouse epithelial cells), and HepG2 (human hepatoma), assessed by Western blotting with anti-phospho-Smad2 antibodies after 1 h stimulation in full medium. (B) Binding of 125I-TGM1 to ALK5 (TβRI) and TβRII, but not other TGF-β/BMP type I and type II family receptors. EL4 cells were incubated with radio-labeled ligand for 3 h on ice, then crosslinked with BS3/DSS (15 min), lysed, and incubated overnight with receptor specific antibodies; finally, complexes were isolated with protein A beads (1 h, 4 °C), separated by SDS-PAGE, and detected by autoradiography. (C) Identification of ~80 kDa coreceptor cross-linked to 125I-D4/5 on the cell surface of 771, EL, and NM18 cells, but not HepG2 cells, as detected by SDS-PAGE and autoradiography. (D) Mass spectrometric identification of CD44 from NM18 cells exposed to biotinylated TGM1 D4/5 and coprecipitated with neutravidin beads. Fold change is calculated compared to control cells that have undergone the same procedure, but without the addition of the TGM1-D4/5-biotin. (E) Immunoprecipitation of iodinated full-length TGM1, and D4/5, but not D1/2/3/4 or D5 alone, with anti-CD44 antibody. Intact cells were first incubated with radiolabeled ligands, crosslinked and washed, and thereafter, cell lysates are prepared, as the input into immunoprecipitation protocols. (F) Coprecipitation of TGF-β receptor I (ALK5) and TGFβRII from MFB-F11 cells by biotinylated D1/2/3, and CD44 by biotinylated D4/5, with Streptavidin beads and analysis by SDS- PAGE and Western blot with anti-TβRI, anti-TβRII and anti-CD44 as indicated. For panels A, B, C, and F, Mol.wt markers are shown in kDa on the left-hand side.

Article Snippet: Recombinant Human CD44 Fc Chimera protein (art# 3660- CD- 050) and Recombinant Mouse CD44 Fc Chimera protein (art# 6127- CD050) were purchased from R&D system (Minneapolis, USA).

Techniques: Western Blot, Binding Assay, Incubation, Labeling, Isolation, SDS Page, Autoradiography, Control, Immunoprecipitation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CD44 acts as a coreceptor for cell-specific enhancement of signaling and regulatory T cell induction by TGM1, a parasite TGF-β mimic.

doi: 10.1073/pnas.2302370120

Figure Lengend Snippet: Fig. 3. Isothermal calorimetry (ITC) confirmation of TGM1 domains 4/5 binding to murine and human CD44. Integrated heats for each of the sequential injections of mCD44 into TGM1-FL (A), TGM1-D1/2/3 (B), TGM1-D4/5 (C), or hCD44 into TGM1-FL (D), TGM1-D1/2/3 (E), TGM1-D4/5 (F) performed in duplicate (blue and purple shading). Integrated heats (y axis) are fit to a 1:1 binding model as a function of CD44:TGM molar ratio (x axis) are shown by the smooth continuous lines (blue and purple shading for the duplicate data sets). Error bars indicate bias in the Nitpic program (24) estimation of the integrated heats; the residuals in the fits are shown in the lower portion of each plot.

Article Snippet: Recombinant Human CD44 Fc Chimera protein (art# 3660- CD- 050) and Recombinant Mouse CD44 Fc Chimera protein (art# 6127- CD050) were purchased from R&D system (Minneapolis, USA).

Techniques: Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CD44 acts as a coreceptor for cell-specific enhancement of signaling and regulatory T cell induction by TGM1, a parasite TGF-β mimic.

doi: 10.1073/pnas.2302370120

Figure Lengend Snippet: Fig. 4. Modulation of CD44 expression controls response to TGM1. (A) SMAD2 phosphorylation of murine NM18 cells with intact (Control) or deleted (KO) CD44 gene expression in response to TGF β and TGM1. Cells were stimulated with 0.5 ng/mL TGF-β or 10 ng/mL TGM1 and lysates were analyzed by SDS-PAGE and Western blotting with anti-CD44, anti-pSMAD2 and anti-GAPDH. Two replicate clones of 2 KO guides were tested. Mol.wt markers are shown in kDa on the left-hand side. (B) Response of NM18-CAGA-mCHERRYd2 transcriptional reporter cells with CD44 KO induced by 0.5 ng/mL TGF-β and 10 ng/mL TGM1. (C and D) Response of HEK293-CAGA-mCHERRYd2 transcriptional reporter cells stably transduced with doxycycline-inducible murine (C) or human (D) CD44, stimulated with TGF-β or the indicated doses of TGM1. (E) Schematic of mouse and human CD44 domain swap constructs. ECD, Extracellular Domains; TM, Transmembrane; ICD, Intracellular Domains. The sequences used are mouse CD44, NP_001034240.1; human CD44, NP_001001391.1. Note that the TM domain is fully conserved between mouse and human. (F) Response of HEK293T-CAGA-mCherry cells stably transduced with doxycycline-inducible mouse and human CD44 domain constructs. (G) Effect of human (hCD44-Fc) or mouse (mCD44-Fc) recombinant CD44-Fc fusion proteins on the response of NIH3T3-CAGA-dynGFP cells to TGM1 (Left) or TGFβ (Right). Ligands were preincubated with control Fc or CD44-Fc for 30 min before adding to cells and fluorescence measured by IncuCyte with Incucyte software.

Article Snippet: Recombinant Human CD44 Fc Chimera protein (art# 3660- CD- 050) and Recombinant Mouse CD44 Fc Chimera protein (art# 6127- CD050) were purchased from R&D system (Minneapolis, USA).

Techniques: Expressing, Phospho-proteomics, Control, Gene Expression, SDS Page, Western Blot, Clone Assay, Stable Transfection, Transduction, Construct, Recombinant, Fluorescence, Software

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: Increased angiogenesis in CD44-null mice. ( A ) Angiogenesis was analysed in C3H, C57BL/6, and in wild-type and Cd44 −/− mice of mixed genetic backgrounds (WT mix and Cd44KO mix, respectively). Basement membrane extract–filled angioreactors containing premixed FGF-2, VEGF and heparin or PBS for uninduced controls were implanted SC into the flanks of mice. Each mouse received 2 angioreactors, 1 per flank. 14 days after implantation, angioreactors were resected and the population of ECs within the angioreactor matrix was assessed by FITC-lectin staining. The number of fluorescent cells was quantitated by microplate reader. Raw readings from independent experiments were scaled by dividing by their quadratic mean. N = 2–5 mice per condition from 2 independent experiments. P value is from Student’s t-test. ( B ) Angiogenesis in Cd44 −/− mice and their heterozygous ( Cd44 +/− ) and wild-type ( Cd44 +/+ ) littermate that had been backcrossed six generations to the C57BL/6 background. The data are represented as the mean ± SEM. Each dot represents the mean of two angioreactors for an individual mouse. N = 8 mice per condition from 2 independent experiments. P values are from ANOVA post hoc comparisons using the Tukey HSD test.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques: Membrane, Staining

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: Recombinant CD44-3MUT Fc fusion protein inhibits angiogenesis in vivo . First, the assay and quantitation were performed similarly to those described in , except that each mouse received 4 angioreactors, 2 per flank. The day after implantation, the mice started to receive CD44-3MUT-Fc, control (rhIgG1-Fc) or vehicle (PBS) every second day via IP injections for 14 days. ( A ) Schematic presentation of experimental design. ( B ) Relative blood vessel invasion into matrix–filled angioreactors. The data are represented as the mean ± SEM. Datapoints show the mean of 4 angioreactors for individual mice. N – the number of independent experiments. GF – growth factors (FGF-2/VEGF). ( C ) Effect size with 95% confidence intervals (upper row) and P values (lower row) of pairwise comparisons of the data shown in ( B ). Effect sizes were calculated by Cohen’s d formula. Confidence intervals were derived using bootstrap resampling. P values are from t tests using pooled SD.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques: Recombinant, In Vivo, Quantitation Assay, Control, Derivative Assay

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: Soluble CD44 concentrations in mouse serum. Blood was collected from mice of different genetic backgrounds and the mice used in the angiogenesis experiments shown in and . ( A ) Serum levels of soluble CD44 in mice from different strains. Each dot represents an individual mouse. Cross indicates the mean. P values are from ANOVA post hoc comparisons using the Tukey HSD test. ( B ) The correlation between relative blood vessel invasion and post-experiment serum sCD44 in wild-type mice. Cd44 -null mice and nude mice were excluded from the dataset. Pearson’s r and the associated P values are shown. ( C ) Post-experiment serum levels of sCD44 in nude mice from different treatment groups. Treatments where more than five mice were analysed are shown. Each dot represents an individual mouse. Cross indicates the mean. GF – growth factors (FGF-2/VEGF). ( D ) The correlation between relative blood vessel invasion and post-experiment serum sCD44 in nude mice. Pearson’s r and the associated P values are shown. In ( B and D ), dashed line is the linear model fit, gray shading is the standard error interval of fitted values.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques:

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet:

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: CD44-3MUT-Fc inhibits EC growth. ( A ) Real-time track of cell adhesion and synchronisation of HUVECs seeded onto 96-well electrode arrays. After seeding, the cells were grown for about 24 h. After that the cells were starved overnight in the media supplemented with 1% FBS (gray area). 1 h before the release from serum starvation (vertical dashed line) the cells were preincubated with different concentrations of rhIgG-Fc or CD44-3MUT-Fc in 5% FBS–containing media. ( B ) Growth curves of HUVECs released from serum starvation by supplementing preincubation media with 25 ng/ml VEGF. Facet labels show rhIgG-Fc or CD44-3MUT-Fc concentrations during preincubation. The data are represented as the mean ± SEM. N – the number of independent experiments. ( C–F ) HUVECs were synchronised and pretreated as in panel A. After preincubation, the cells were stimulated either with 25 ng/ml FGF-2 ( C ), 25 ng/ml VEGF ( D ), 63 ng/ml HGF ( E ) or 10 ng/ml GDF-2 ( F ). After 72 h, the number of viable cells was quantitated by measuring the ATP per well. Left: the effect of growth factor stimulation and the effect of 10 nM fumagillin (FUM) as a positive control for inhibition of cell proliferation. Right: the dose-response curves of rhIgG-Fc (filled triangles) and CD44-3MUT-Fc (filled circles). The data are represented as the mean ± SEM. N = 3–4 independent experiments. ( G ) CD44-3MUT-Fc dose-response curves for FGF-2, GDF-2, HGF or VEGF stimulated HUVEC. The data are represented as the mean ± SEM. ( H ) Apoptosis of HUVECs stimulated with different growth factors and treated with 12.64 μM (-4.9 log 10 M) rhIgG-Fc, CD44-3MUT-Fc or left untreated. Apoptosis was quantitated by Annexin V staining. The data are represented as the mean ± SEM. N = 2 independent experiments. P values are from the ANOVA post hoc comparisons using the Tukey HSD test.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques: Positive Control, Inhibition, Staining

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: CD44 knockdown augments EC growth. siRNA transfected HUVECs were plated onto 96-well electrode arrays. After 24 h, the cells were starved in 1% FBS media overnight. After starving, the cells were released from cell cycle block by the addition of 20% FBS (A) ; 8 ng/ml, 25 ng/ml, or 79 ng/ml FGF-2 or VEGF (C) ; and 2.5 ng/ml or 10 ng/ml GDF-2 (E) . Following stimulation, HUVEC growth was monitored by recording electrode impedance. Raw impedance readings are shown to allow direct comparison to endpoint measurements. N = 2 for 5% and 20% FBS, 25 ng/ml and 79 ng/ml FGF-2, and 8 ng/ml and 25 ng/ml VEGF. (B, D, F) 72 h after release from cell cycle block the viable cell numbers were determined by measuring the ATP per well. Treatments are labeled as shown in ( B ). The data are represented as the mean ± SEM. P values are from the ANOVA post hoc comparisons using the Tukey HSD test. P values ≤ 0.05 of siCD44-siNTP comparisons are shown. siCD44-siNTP comparisons: N = 4 for 2.5 ng/ml and 10 ng/ml GDF-2, and N = 5 for 20% FBS, 25 ng/ml and 79 ng/ml FGF-2, and 8 ng/ml and 25 ng/ml VEGF. (G) Western blot analysis of CD44 silencing in HUVECs transfected with 30 nM siRNAs for 48 h. siNTP – non-targeting siRNA pool, siVIM – vimentin-targeting pool, siCD44 – CD44-targeting pool, UT – non-transfected cells.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques: Knockdown, Transfection, Blocking Assay, Comparison, Labeling, Western Blot

Journal: PLoS ONE

Article Title: CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site

doi: 10.1371/journal.pone.0138137

Figure Lengend Snippet: The protein backbone is represented as a blue cartoon. The HA 8-mer is shown as pink sticks. Disulfide bonds are shown as yellow sticks and the cysteine residues from which they are formed are numbered according to their position in the mouse CD44 sequence (human numbering is shown in parenthesis). (B) For each disulfide bond dark grey bars show solvent accessibility and light grey bars show bond strain energy calculated from the PDB coordinates (PDB ID:2JCQ for mouse and 1UUH for human, the coordinates for HA were removed from 2JCQ prior to analysis) using ( http://powcs.med.unsw.edu.au/research/adult-cancer-program/services-resources/disulfide-bond-analysis-tool/disulfide-bond ).

Article Snippet: Human and mouse CD44 Fc proteins were purchased from R&D systems.

Techniques: Sequencing, Solvent

Journal: PLoS ONE

Article Title: CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site

doi: 10.1371/journal.pone.0138137

Figure Lengend Snippet: (A) hCD44-Fc and (B) mCD44-Fc binding to plates coated with 50 μg/ml HA. Columns represent non reduced control CD44-Fc (dark grey) and TCEP-HCl reduced CD44-Fc (light grey). Mean and SEM of peptide chromatographic areas from three experiments are shown, the differences between non reduced and TCEP-HCl reduced chimera: *, P < 0.05; **, P < 0.01. Titrations of Trx1 reduced (C) human CD44-Fc and (D) mouse CD44-Fc binding to plates coated with 50 μg/ml HA. For both mouse and human CD44-Fc titrations the binding curves represent non reduced control CD44-Fc (black circles) and Trx1 reduced CD44-Fc (black squares). Binding curves are constructed from data of three independent experiments and equilibrium dissociation constants were determined by fitting to a single binding site model.

Article Snippet: Human and mouse CD44 Fc proteins were purchased from R&D systems.

Techniques: Binding Assay, Control, Construct

Journal: PLoS ONE

Article Title: CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site

doi: 10.1371/journal.pone.0138137

Figure Lengend Snippet: Light grey trace is isotype control, black trace is anti-CD44. (C) Non reduced SDS-PAGE gel showing reduced proteins on the surface of hCD44 transfected CHO cells after reduction (green bands) overlaid with a western blot of CD44 (red). (D) non reduced SDS-PAGE gel of eluted proteins which contain reduced disulfide bonds (green bands) from a CD44 immunoprecipitation of hCD44 transfected CHO cells that had been reduced with either TCEP-HCl or Trx1.

Article Snippet: Human and mouse CD44 Fc proteins were purchased from R&D systems.

Techniques: Control, SDS Page, Transfection, Western Blot, Immunoprecipitation