Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Table of Sequences for Forward and Reverse Primers for Genes Analyzed by PCR

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques:

Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Nodal, but not Cripto, is expressed in adult human islets. Expression of Nodal and Cripto mRNA and protein in isolated adult human islets are shown. A, PCR for Nodal, Cripto, and activin receptors using 0.8 μg of mRNA and specific primers (Table 1) resolved on a 2% agarose gel. HPNE and PANC-1 cells were used as controls. B, Western blot for Nodal protein in isolated human islets cultured for 24 hours before islets were harvested, washed with PBS, and frozen at −80°C. C, Western blot for Cripto in isolated human islets, with MCF7 cells as a positive control.

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques: Expressing, Isolation, Agarose Gel Electrophoresis, Western Blot, Cell Culture, Positive Control

Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Nodal stimulates human β-cell proliferation while inhibiting α-cell proliferation. Flow cytometry analysis of human islets trypisinized to single cells after 120 hours of exposure to Cripto (250 ng/mL) and/or Nodal (10 μg/mL). Cells were stained for insulin, glucagon, and BrdU to evaluate for proliferating β- and α-cells in Cripto- and/or Nodal-treated groups compared with controls. A, Gating strategy used for flow cytometry analysis. Viable cells, cells that did not take up Live/Dead stain, were gated for insulin and glucagon positive cells (red arrow). Endocrine cells were further gated for double-positive insulin +/BrdU+ (blue arrow) or glucagon+/BrdU+ (green arrow), indicating the proliferating subset of β and α-cells. (BrdU isotype is shown.) B, Comparison of proliferating β-cells in Cripto- and/or Nodal-treated islets compared with control. A total of 3773 ± 577 β-cells were counted per treatment group per experiment (n = 5). C, Comparison of proliferating α-cells in Cripto- and/or Nodal-treated islets compared with control. A total of 5707 ± 961 α-cells were counted per treatment group per experiment (n = 4). Bars indicate mean ± SEM. *, P < .05.

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques: Flow Cytometry, Staining, Comparison, Control

Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Measurement of β-cell proliferation by immunofluorescence confirms robustness of flow cytometry proliferation assay. A, Example of control and Nodal-treated (10 μg/mL) human islets embedded in paraffin, sectioned, and stained for insulin (green), BrdU (red), and DAPI (blue). β-Cells were considered BrdU+ if BrdU and DAPI colocalized and insulin staining surrounded the nucleus (white arrow). B, Quantification of BrdU-positive β-cells in islets after 120 hours exposure to Cripto 250 ng/mL, Nodal 10 μg/ml, or Nodal (10 μg/mL) + Cripto (250 ng/mL) compared with control. Insulin+/BrdU+ cells were quantified per total number of insulin+ cells and results are shown as a percentage of control [n = 4 separate experiments (mean number of β-cells counted per group per experiment: control, 3359 ± 265; Cripto, 3074 ± 253; Nodal, 3137 ± 414; Nodal + Cripto, 3628 ± 280]. C, Quantification of Ki67-positive β-cells in islets after 120 hours exposure to Nodal 10 μg/mL compared with control. Insulin+/Ki67+ cells were quantified per total number of insulin+ cells and results are shown as a percentage of control [n = 3 separate experiments (mean number of β-cells counted per group per experiment: control, 4245 ± 73; Nodal, 4247 ± 175]. D, Example of control and Nodal-treated (10 μg/ml) human islets stained for insulin (green), Ki67 (red), and DAPI (blue). β-Cells were considered Ki67+ if Ki67 and DAPI colocalized and insulin staining surrounded the nucleus (white arrow). Bars indicate mean ± SEM. Scale bar, 50 μM. *, P < .05.

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques: Immunofluorescence, Flow Cytometry, Proliferation Assay, Control, Staining

Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Nodal signals through SMAD proteins in adult human islets, whereas Nodal and Cripto have no effect on acute or chronic AKT or MAPK activity. Western blot analysis of cell signaling pathways in human islets exposed to acute (30 min) treatment with Nodal (10 μg/mL) and/or Cripto (250 ng/mL). A, Phosphorylated-SMAD2 (n = 5). B, Phosphorylated AKT (n = 3) and phosphorylated ERK 1/2 (n = 4). Western blot analysis of cell signaling pathways in human islets exposed to chronic (120 h) treatment with Nodal (10 μg/mL) and/or Cripto (250 ng/mL). C, Phosphorylated-AKT and ERK 1/2 (n = 3–4). Bars indicate mean ± SEM. *, P < .05.

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques: Activity Assay, Western Blot, Protein-Protein interactions

Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Chronic Nodal and Cripto treatment does not enhance dedifferentiation of human islet cells. Western blot and immunofluorescent analysis of markers of differentiation and cell signaling pathways in human islets were conducted at baseline compared with untreated cultured islets (control) and islets exposed to chronic (120 h) treatment with Nodal (10 μg/mL) and/or Cripto (250 ng/mL). A, Representative images of CK19 expression in baseline, control, and treated islets after 120 hours of culture. CK19 (red), insulin (green), and DAPI (blue) are shown. B, CK19 and SOX9 expression in baseline islets compared with control and treated islets after 120 hours culture (n = 3 for both). Bars indicate mean ± SEM.

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques: Western Blot, Protein-Protein interactions, Cell Culture, Control, Expressing

Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Nodal and Cripto do not adversely affect viability or stimulate apoptosis in human islets. A, Viability of human islet cells by flow cytometry assay (Live/Dead) after 120 hours of exposure to Nodal (10 μg/mL) and/or Cripto (250 ng/mL) compared with control. Live cells are shown within the gate (n = 5). B, TUNEL staining of human islets exposed to Nodal (10 μg/mL) and Cripto (250 ng/mL) for 120 hours compared with control (n = 3). Arrows indicate apoptotic cells (as indicated by TUNEL+/DAPI+). Arrowheads indicate examples of nonspecific fluorescence (DAPI−) that were not counted as apoptotic cells. Then 49 ± 1.4 islets were counted per treatment group per experiment. Bars indicate mean ± SEM. Scale bar, 50 μM. C, Western blot for cleaved caspase-3 expression in islets cultured for 120 hours exposed to Cripto (250 ng/mL) and/or Nodal (10 μg/mL) compared with control. Human islets exposed to staurosporine (STS) 0.5 μM for 18 hours were used as positive control. Image shown is representative of three separate experiments.

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques: Flow Cytometry, Control, TUNEL Assay, Staining, Fluorescence, Western Blot, Expressing, Cell Culture, Positive Control