Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: Traction forces are necessary for definitive endoderm specification. Mouse ESCs (CCE and R1) were grown on decellularized fibroblast-derived ECM in definitive endoderm induction medium, with or without 10 μM blebbistatin. (A) Immunofluorescent images of SOX17 (green) and Hoechst 33342 (blue)-stained nuclei were taken after 5 days and (B) the mean±s.e.m. (n>300) nuclear intensity of SOX17 was quantified. *P<0.05. Scale bar: 20 µm. a.u., arbitrary units.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 μg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 μM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay, Staining

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: Cell contractility is activated upon definitive endoderm induction. Mouse ESCs (CCE and R1) were grown in either definitive endoderm (DE) or pluripotency (PP) induction medium on decellularized fibroblast-derived ECM containing FRET-FN. (A) Confocal z-stacks of the FRET fibronectin matrix were captured after 0 (No Cells) and 5 days of induction and (B) the average FRET intensity ratio of the ESC associated matrix was quantified (n>16). ESCs were treated with 50 μM blebbistatin for 1 h at the end of definitive endoderm or pluripotency induction and (C) the average FRET intensity ratios of ESC-associated matrix (n>16) and (D) SOX17 gene expression were quantified (mean±s.e.m., n=3). For boxplots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90th percentiles. *P<0.05.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 μg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 μM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay, Gene Expression

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: ECM signaling regulates definitive endoderm induction and contractility. (A) Mouse ESCs (CCE and R1) were grown in definitive endoderm (DE) or pluripotency (PP) induction medium on decellularized fibroblast-derived matrices supplemented with FRET fibronectin and either 0 μg/ml (– exogenous laminin) or 50 μg/ml (+ exogenous laminin) mouse laminin-111. Equal amounts of DOC-insoluble ECM were blotted for laminin or fibronectin after 1 and 5 days of ESC culture. (B) R1 ESC mean±s.e.m. (n>300) nuclear SOX17 staining intensity and (C) average extracellular matrix FRET intensity ratios (the box represents the 25–75th percentiles, and the median is indicated; the whiskers show the 10–90th percentiles, n>10) were measured after 5 days of definitive endoderm induction in the presence or absence of the α5β1-integrin function-blocking antibody BIIG2. *P<0.05. a.u., arbitrary units.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 μg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 μM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay, Staining, Blocking Assay

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: Laminin effects on contractility are specific to definitive endoderm. After 5 days in either definitive endoderm (DE) or pluripotency (PP) induction medium, and the average FRET intensity ratios of ESC-associated matrix were quantified (the box represents the 25–75th percentiles, and the median is indicated; the whiskers show the 10–90th percentiles) for mouse ESCs (CCE and R1) grown on decellularized fibroblast-derived ECM containing FRET-FN and either 0 μg/ml (– laminin) or 50 μg/ml (+ laminin) exogenous laminin. n>16. *P<0.05.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 μg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 μM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: Traction forces are necessary for definitive endoderm specification. Mouse ESCs (CCE and R1) were grown on decellularized fibroblast-derived ECM in definitive endoderm induction medium, with or without 10 μM blebbistatin. (A) Immunofluorescent images of SOX17 (green) and Hoechst 33342 (blue)-stained nuclei were taken after 5 days and (B) the mean±s.e.m. (n>300) nuclear intensity of SOX17 was quantified. *P<0.05. Scale bar: 20 µm. a.u., arbitrary units.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 µM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay, Staining

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: Cell contractility is activated upon definitive endoderm induction. Mouse ESCs (CCE and R1) were grown in either definitive endoderm (DE) or pluripotency (PP) induction medium on decellularized fibroblast-derived ECM containing FRET-FN. (A) Confocal z-stacks of the FRET fibronectin matrix were captured after 0 (No Cells) and 5 days of induction and (B) the average FRET intensity ratio of the ESC associated matrix was quantified (n>16). ESCs were treated with 50 μM blebbistatin for 1 h at the end of definitive endoderm or pluripotency induction and (C) the average FRET intensity ratios of ESC-associated matrix (n>16) and (D) SOX17 gene expression were quantified (mean±s.e.m., n=3). For boxplots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90th percentiles. *P<0.05.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 µM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay, Expressing

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: ECM signaling regulates definitive endoderm induction and contractility. (A) Mouse ESCs (CCE and R1) were grown in definitive endoderm (DE) or pluripotency (PP) induction medium on decellularized fibroblast-derived matrices supplemented with FRET fibronectin and either 0 μg/ml (– exogenous laminin) or 50 μg/ml (+ exogenous laminin) mouse laminin-111. Equal amounts of DOC-insoluble ECM were blotted for laminin or fibronectin after 1 and 5 days of ESC culture. (B) R1 ESC mean±s.e.m. (n>300) nuclear SOX17 staining intensity and (C) average extracellular matrix FRET intensity ratios (the box represents the 25–75th percentiles, and the median is indicated; the whiskers show the 10–90th percentiles, n>10) were measured after 5 days of definitive endoderm induction in the presence or absence of the α5β1-integrin function-blocking antibody BIIG2. *P<0.05. a.u., arbitrary units.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 µM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay, Staining, Blocking Assay

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: Laminin effects on contractility are specific to definitive endoderm. After 5 days in either definitive endoderm (DE) or pluripotency (PP) induction medium, and the average FRET intensity ratios of ESC-associated matrix were quantified (the box represents the 25–75th percentiles, and the median is indicated; the whiskers show the 10–90th percentiles) for mouse ESCs (CCE and R1) grown on decellularized fibroblast-derived ECM containing FRET-FN and either 0 μg/ml (– laminin) or 50 μg/ml (+ laminin) exogenous laminin. n>16. *P<0.05.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 µM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay