Journal: Scientific reports

Article Title: Non-invasive detection of DNA methylation states in carcinoma and pluripotent stem cells using Raman microspectroscopy and imaging.

doi: 10.1038/s41598-019-43520-z

Figure Lengend Snippet: Figure 3. Investigation of global DNA methylation changes. (a) 5mC expression in mESCs cultured under control conditions or in 2i medium. 2i medium-adapted cells show a significantly lower DNA methylation. Two-tailed t-test, n = 3. (b) Immunofluorescence staining of 5mC. Scale bars equal 20 µm. (c + d) Quantitative fluorescence intensity analysis of 5mC-stained mESCs. 2i medium-cultured mESCs show a significant lower mean intensity and less 5mC-foci/cell (indicated by white arrows in b) when compared with cells cultured in the presence of serum (control). Two-tailed t-test, n = 3.

Article Snippet: CCE mESCs (ATCC® SCRC-1023TM) were cultured in KO-DMEM (Thermo Fisher Scientific), supplemented with 15% fetal bovine serum, 1% Penicillin-Streptomycin, 1% MEM non-essential amino acids, 1% L-Glutamine, 0.2% HEPES, 0.1% 2-Mercaptoethanol (all Thermo Fisher Scientific) and 0.1% Leukemia inhibitory factor (Merck, Darmstadt, Germany).

Techniques: DNA Methylation Assay, Expressing, Cell Culture, Control, Two Tailed Test, Immunofluorescence, Staining, Fluorescence

Journal: Scientific reports

Article Title: Non-invasive detection of DNA methylation states in carcinoma and pluripotent stem cells using Raman microspectroscopy and imaging.

doi: 10.1038/s41598-019-43520-z

Figure Lengend Snippet: Figure 4. Identification of mESC Raman signatures due to an altered global DNA methylation. (a) Raman spectra of mESCs cultured in (i) control and (ii) 2i medium. Raman signatures that can be assigned to DNA bands are indicated by black arrows. The standard deviation is indicated by the gray lines. (b) Statistical analysis of significantly increased Raman bands, which can be assigned to DNA methylation. Two-tailed t-test, n = 85. (c) Score plot of PC 1 and PC 3 shows a grouping tendency of mESCs cultured in control and 2i media. The direction of the gray arrow indicates the loss of DNA methylation. The score mean value of both groups for PC 4 differs significantly from each other. Two-tailed t-test, n = 85 (d) PC 4 loading describes the Raman shifts that vary in 2i-adapted mESCs compared with mESCs cultured in the control medium. Peaks of interest are indicated by black arrows.

Article Snippet: CCE mESCs (ATCC® SCRC-1023TM) were cultured in KO-DMEM (Thermo Fisher Scientific), supplemented with 15% fetal bovine serum, 1% Penicillin-Streptomycin, 1% MEM non-essential amino acids, 1% L-Glutamine, 0.2% HEPES, 0.1% 2-Mercaptoethanol (all Thermo Fisher Scientific) and 0.1% Leukemia inhibitory factor (Merck, Darmstadt, Germany).

Techniques: DNA Methylation Assay, Cell Culture, Control, Standard Deviation, Two Tailed Test

Journal: Scientific reports

Article Title: Non-invasive detection of DNA methylation states in carcinoma and pluripotent stem cells using Raman microspectroscopy and imaging.

doi: 10.1038/s41598-019-43520-z

Figure Lengend Snippet: Figure 5. Comparison of Raman spectra from mouse pluripotent stem cells cultured in either control or 2i medium with human colon cancer WT and DNMT−/− cells. (a) 3D score plot of Raman spectra from control mESCs, 2i mESCs as well as human colon cancer WT and DNMT1−/− cells showing PC 2, PC 4 and PC 5. There is a grouping tendency between control mESCs and WT cells (blue shades), as well as 2i mESCs, DNMT1−/− cells (green shades). (b) The grouping tendency is visualized by a box plot of PC 5 score values. It shows significantly lower scores for control mESCs and WT cells when compared with 2i mESC and DNMT1−/− cells. One-way ANOVA, n = 30.

Article Snippet: CCE mESCs (ATCC® SCRC-1023TM) were cultured in KO-DMEM (Thermo Fisher Scientific), supplemented with 15% fetal bovine serum, 1% Penicillin-Streptomycin, 1% MEM non-essential amino acids, 1% L-Glutamine, 0.2% HEPES, 0.1% 2-Mercaptoethanol (all Thermo Fisher Scientific) and 0.1% Leukemia inhibitory factor (Merck, Darmstadt, Germany).

Techniques: Comparison, Cell Culture, Control

Journal: Scientific reports

Article Title: Non-invasive detection of DNA methylation states in carcinoma and pluripotent stem cells using Raman microspectroscopy and imaging.

doi: 10.1038/s41598-019-43520-z

Figure Lengend Snippet: Figure 6. Detection of 5mC-foci with high-resolution Raman imaging of HCT116 cells and mESCs. (a) Anti- 5mC immunofluorescence staining of HCT116 WT and DNMT1−/− cells. (b) Heat maps of the Raman images obtained from HCT116 WT and DNMT1−/− cells using the sum intensities of 1257 cm−1, 1379 cm−1 and 1579 cm−1. (c) Average spectra from high-intensity regions and low-intensity regions of the heat maps from panel (b). (d,h) Statistical analysis of the Raman images from panels b and f. Two-tailed t-test, n = 3. (e) Anti- 5mC immunofluorescence staining of mESC control and 2i mESCs. (f) Heat maps of the Raman images from mESC control and 2i mESCs using the sum intensities described in panel b. (g) Average spectra from high- intensity regions and low-intensity regions of the heat maps from panel f. Scale bars in all images equal 5 µm.

Article Snippet: CCE mESCs (ATCC® SCRC-1023TM) were cultured in KO-DMEM (Thermo Fisher Scientific), supplemented with 15% fetal bovine serum, 1% Penicillin-Streptomycin, 1% MEM non-essential amino acids, 1% L-Glutamine, 0.2% HEPES, 0.1% 2-Mercaptoethanol (all Thermo Fisher Scientific) and 0.1% Leukemia inhibitory factor (Merck, Darmstadt, Germany).

Techniques: Imaging, Immunofluorescence, Staining, Two Tailed Test, Control

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