Journal: Stem cells (Dayton, Ohio)

Article Title: Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin.

doi: 10.1634/stemcells.2008-0291

Figure Lengend Snippet: Figure 2. Human embryonic stem cell integrin function in initial substrate adhesion. (A): HUES1 cell attachment dose-response binding curve to various natural matrices. (B): Functional validation of integrin binding capacity on various matrices using integrin-specific blocking Abs. , p .01. Data from at least three independent experiments were collected and are displayed as average SEM. Abbreviations: Ab, antibody; BSA, bovine serum albumin; Col IV, collagen IV; E, entactin; FN, fibronectin; MG, Matrigel; mLN, mouse laminin; VN, vitronectin.

Article Snippet: For feeder-free defined cultures, all three lines were cultured in mTeSR1 (StemCell Technologies, Vancouver, BC, Canada, http:// www.stemcell.com) on recombinant vitronectin (5 ng/ l) (R&D Systems Inc., Minneapolis, http://www.rndsystems.com).

Techniques: Cell Attachment Assay, Binding Assay, Functional Assay, Biomarker Discovery, Blocking Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin.

doi: 10.1634/stemcells.2008-0291

Figure Lengend Snippet: Figure 3. Human embryonic stem cell ex- pansion in various media is matrix-dependent. HUES1 cells were trypsinized and replated in triplicate in 96-well plates on various matrices. Cell proliferation was measured quantitatively at different time points. (A): HUES1 cell ex- pansion on various substrates in MEF-CM. (B): HUES1 cell expansion on various sub- strates in mTeSR1 chemically defined me- dium. (C–F): HUES1 cell expansion assays in the presence or absence of integrin 5 and 6 blocking antibodies, which were added 4 hours postplating to circumvent differential attach- ment on, LNE (C), FN (D), collagen IV (E), and vitronectin (F). Experiments were re- peated at least three times, and one represen- tative experiment is shown here. Each time point represents the mean absorbance of a trip- licate SEM. Abbreviations: ColIV, collagen IV; FN, fibronectin; h, hours; LNE, lami- nin entactin; MEF-CM, mouse embryonic fibroblast feeder-conditioned medium; MG, Matrigel; pVN, plasma vitronectin.

Article Snippet: For feeder-free defined cultures, all three lines were cultured in mTeSR1 (StemCell Technologies, Vancouver, BC, Canada, http:// www.stemcell.com) on recombinant vitronectin (5 ng/ l) (R&D Systems Inc., Minneapolis, http://www.rndsystems.com).

Techniques: Blocking Assay, Clinical Proteomics

Journal: Stem cells (Dayton, Ohio)

Article Title: Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin.

doi: 10.1634/stemcells.2008-0291

Figure Lengend Snippet: Figure 4. Recombinant VN supports human embryonic stem cells in mTeSR1. (A): HUES1 cell expansion assay on pVN and recombinant VN. (B): HUES1 cell attachment in the presence and absence of integrin V5-blocking Abs on both pVN and recombinant VN. (C–E): Representative bright-field pictures of HUES1, HES2, and HESC-NL3 cultured for eight passages on recombinant VN in mTeSR1. (F): Karyogram of HESC-NL3 cultured for seven passages on recombinant VN in mTeSR1 showing a normal diploid 46,xy karyotype. (G–L): Immunostaining for stem cell markers and DNA for HESC-NL3 (overlay). (G): E-Cad. (H): GCTM2. (I): Oct3/4a. (J): SOX2. (K): SSEA3. (L): SSEA4. (M–O): Immunostaining for HESC-NL3 differentiation markers representing the three germ layers: AFP (endoderm) (M), III tubulin (ectoderm) (N), and -actinin (mesoderm) (O). Scale bars 50 m. Abbreviations: Ab, antibody; AFP, -fetoprotein; BSA, bovine serum albumin; E-Cad, E-cadherin; pVN, plasma vitronectin; VN, vitronectin.

Article Snippet: For feeder-free defined cultures, all three lines were cultured in mTeSR1 (StemCell Technologies, Vancouver, BC, Canada, http:// www.stemcell.com) on recombinant vitronectin (5 ng/ l) (R&D Systems Inc., Minneapolis, http://www.rndsystems.com).

Techniques: Recombinant, Cell Attachment Assay, Blocking Assay, Cell Culture, Immunostaining, Clinical Proteomics

Journal: PLoS ONE

Article Title: Chemokine (C-C Motif) Ligand 20, a Potential Biomarker for Graves' Disease, Is Regulated by Osteopontin

doi: 10.1371/journal.pone.0064277

Figure Lengend Snippet: (A) Induction of CCL20 level in plasma from uGD patients and healthy controls. Medium: RPMI 1640; NP, normal plasma; NP+ OPN: normal plasma added with 0.1 µg/ml OPN; PP, patient plasma; PP+OPNmAb : patient plasma pretreated with 5 µg/ml OPN-neutralizing antibody for 30 min. *, P <0.05 versus medium control; #, P <0.05 versus Ctrl mAb. (B, C) OPN induced CCL20 mRNA expressions (B) and protein levels (C) in a time and dose-dependent manner. For time course, the PBMCs were cultured in the presence of human recombinant OPN (1 µg/ml) and analyzed at the indicated time points. To determine the dose dependent manner of CCL20 expression, the PBMCs were cultured for 12 hours with OPN at the indicated concentrations. CD4+T cells and culture medium were then purified and analyzed. Shown are representative results from 3 independent experiments with separate specimens. All data were presented as mean±SEM. *, P <0.05; **, P <0.01, versus medium control.

Article Snippet: For blocking experiments, purified antibodies against human OPN (Human OPN Affinity Purified Polyclonal Ab, AF1433, 5µg/ml), human β3 receptor (Human Integrin alpha V beta 3 MAb, MAB3050, 5µg/ml), human IL-17 (Human IL-17 Affinity Purified Polyclonal Ab, AF-317-NA, 1µg/ml) and isotype-matched control antibodies (all purchased from R&D Systems) were added.

Techniques: Cell Culture, Recombinant, Expressing, Purification

Journal: PLoS ONE

Article Title: Chemokine (C-C Motif) Ligand 20, a Potential Biomarker for Graves' Disease, Is Regulated by Osteopontin

doi: 10.1371/journal.pone.0064277

Figure Lengend Snippet: (A) CCL20 gene expressions in isolated CD4+T cells from 8 hCD and 8 uGD in the presence or absence of 1 µg/ml rOPN for 12 h. (B) Expressions of OPN receptors on CD4+ T cells from uGD patients, eGD patients, nGD patients and hCD. (C) β3 integrin receptor antibody blocked induction of CCL20 mRNA and protein levels by OPN. The freshly isolated PBMCs were cultured in the presence or absence of 1 µg/ml rOPN for 12 h. Blocking Abs to integrin β3, or its isotype control mAb (IgG) were added at 5 µg/ml. Supernatants from cultures and CD4+T cells separated from PBMCs were used to analyze the expression levels of CCL20. Shown are representative results from 3 independent experiments with separate specimens. All data were presented as mean±SEM. *, P <0.05; ***, P <0.001, versus medium control; #, P <0.05 versus Ctrl mAb.

Article Snippet: For blocking experiments, purified antibodies against human OPN (Human OPN Affinity Purified Polyclonal Ab, AF1433, 5µg/ml), human β3 receptor (Human Integrin alpha V beta 3 MAb, MAB3050, 5µg/ml), human IL-17 (Human IL-17 Affinity Purified Polyclonal Ab, AF-317-NA, 1µg/ml) and isotype-matched control antibodies (all purchased from R&D Systems) were added.

Techniques: Isolation, Cell Culture, Blocking Assay, Expressing

Journal: PLoS ONE

Article Title: Chemokine (C-C Motif) Ligand 20, a Potential Biomarker for Graves' Disease, Is Regulated by Osteopontin

doi: 10.1371/journal.pone.0064277

Figure Lengend Snippet: (A, B) PBMCs were isolated from normal subjects and treated with rOPN (1 µg/ml) at different time point. The IL-17 mRNA expression in purified CD4+T cells (A) and protein level in culture medium (B) were analyzed. Induction of CCL20 mRNA in CD4+T cells (C) and protein levels in culture medium (D) by OPN was blocked by antibody against IL-17, and inhibitors of IKK and MAPKs. Shown are representative results from 3 independent experiments with separate specimens. All data were presented as mean±SEM. *, P <0.05 versus OPN; # P <0.05 versus medium control; ns>0.05 versus OPN.

Article Snippet: For blocking experiments, purified antibodies against human OPN (Human OPN Affinity Purified Polyclonal Ab, AF1433, 5µg/ml), human β3 receptor (Human Integrin alpha V beta 3 MAb, MAB3050, 5µg/ml), human IL-17 (Human IL-17 Affinity Purified Polyclonal Ab, AF-317-NA, 1µg/ml) and isotype-matched control antibodies (all purchased from R&D Systems) were added.

Techniques: Isolation, Expressing, Purification

Journal: Cell metabolism

Article Title: The nuclear receptor ESRRA protects from kidney disease by coupling metabolism and differentiation

doi: 10.1016/j.cmet.2020.11.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Kidney organoids differentiation ES[4] human embryonic stem cells were grown on vitronectin coated plates (1001–015, Life Technologies).

Techniques: Western Blot, Immunohistochemistry, Immunofluorescence, Plasmid Preparation, Recombinant, Protease Inhibitor, SYBR Green Assay, Cell Culture, Bicinchoninic Acid Protein Assay, Blocking Assay, Knock-Out, Software