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

Article Title: Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells

doi: 10.1073/pnas.0810230105

Figure Lengend Snippet: Effect of NOC-18 and l-NAME in H-9 cells. Human ES cells (H-9) were subjected to differentiation by using the EB method, and the differentiated cells were exposed to an NO donor, NOC-18 (1–2 μM), and a nonspecific NOS inhibitor, l-NAME (2 mM), on day 17 for 24 h. mRNA expression for the indicated genes (MLC2, NkX2.5, sGC α1, and GAPDH) was analyzed by real-time PCR. All of the samples were normalized by using housekeeping gene GAPDH, and data were analyzed by the 2−ΔΔCt method. Error bars (±SEM) indicate significance using paired Student's t test is indicated. *, P < 0.05; **, P < 0.01; n = 4.

Article Snippet: H-9 (WA-09, human ES) were purchased from WiCell Research Institute and grown in 80% DMEM/F12, 20% knockout serum replacer, 1 mM l -glutamine, 0.1 mM β-mercaptoethanol, and 1 mM nonessential amino acids supplemented with 4 ng/ml bFGF on mitotically-inactivated MEF feeder layers and matrigel.

Techniques: Expressing, Real-time Polymerase Chain Reaction

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

Article Title: Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells

doi: 10.1073/pnas.0810230105

Figure Lengend Snippet: Effect of BAY 41-2272 on Nkx2.5 mRNA on H-9 cells. (A and B) Partially differentiated H-9 cells were exposed to a single treatment on day 13 (A) or multiple treatments (starting on day 7, total number of exposures 4 (B). The cells were harvested on day 14, and mRNA expression of Nkx2.5 was determined by real-time PCR. All of the samples were normalized with the housekeeping gene GAPDH. *, P < 0.05; **, P < 0.01; n = 3–6. (C) The protein from differentiated H-9 cells either untreated or treated with BAY 41-2272 (1–3 μM) or ascorbic acid (30–90 μM) on days 7, 9, 11, and 13 and harvested on day 14 was extracted and detected with specific antibodies against MLC2 and β-actin by using Western blot–ECL analysis.

Article Snippet: H-9 (WA-09, human ES) were purchased from WiCell Research Institute and grown in 80% DMEM/F12, 20% knockout serum replacer, 1 mM l -glutamine, 0.1 mM β-mercaptoethanol, and 1 mM nonessential amino acids supplemented with 4 ng/ml bFGF on mitotically-inactivated MEF feeder layers and matrigel.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

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

Article Title: Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells

doi: 10.1073/pnas.0810230105

Figure Lengend Snippet: Effect of NO donors and sGC activators in H-9 cells. Partially differentiated cells were exposed to a single treatment of the NO donor SNAP (50 μM), BAY 41-2272 (5 μM), and the combination of SNAP+BAY41 (A) or multiple treatments of NOC-18 (1 μM), BAY 41-2272 (3 μM), or the combination of NOC-18+BAY41, YC-1 (3 μM) or the combination of NOC-18 + YC-1 (B and C). The samples were analyzed for Nkx2.5 (A and B) and sGC α1 transcript levels (C) by quantitative RT-PCR. Error bars indicate ± SEM. Significance using paired Student's t test is shown: *, P < 0.05; **, P < 0.01. n = 5–7.

Article Snippet: H-9 (WA-09, human ES) were purchased from WiCell Research Institute and grown in 80% DMEM/F12, 20% knockout serum replacer, 1 mM l -glutamine, 0.1 mM β-mercaptoethanol, and 1 mM nonessential amino acids supplemented with 4 ng/ml bFGF on mitotically-inactivated MEF feeder layers and matrigel.

Techniques: Quantitative RT-PCR

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

Article Title: Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells

doi: 10.1073/pnas.0810230105

Figure Lengend Snippet: Immunofluorescence detection of MLC2 and cGMP in H-9 cells exposed to NO donor and sGC activator. Differentiated cells were exposed to NOC-18 (1 μM; Top), BAY 41-2272 (3 μM; Middle), or the combination of the two (C) for 24 h at 37 °C. The cells were fixed with paraformaldehyde and incubated with antibodies to MLC2 and cGMP that was followed by detection with fluorescent-conjugated secondary anti-mouse or rabbit antibodies. (Magnification: 20×.)

Article Snippet: H-9 (WA-09, human ES) were purchased from WiCell Research Institute and grown in 80% DMEM/F12, 20% knockout serum replacer, 1 mM l -glutamine, 0.1 mM β-mercaptoethanol, and 1 mM nonessential amino acids supplemented with 4 ng/ml bFGF on mitotically-inactivated MEF feeder layers and matrigel.

Techniques: Immunofluorescence, Incubation

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

Article Title: Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells

doi: 10.1073/pnas.0810230105

Figure Lengend Snippet: cGMP accumulation in undifferentiated and differentiated H-9 cells. H-9 cells were harvested with 1 mM EDTA in DPBS and incubated for 10 min at 37 °C in DPBS containing 1 mM IBMX with 0.1% DMSO or 5 μM BAY 41-2272 (2–3 × 107 cells per mL) followed by the addition of NO donors. Cells were incubated for 4 min and then 50 μL of ice-cold 1 M perchloric acid was added and cGMP was determined by ELISA. Data are expressed in pmol cGMP per mg of protein.

Article Snippet: H-9 (WA-09, human ES) were purchased from WiCell Research Institute and grown in 80% DMEM/F12, 20% knockout serum replacer, 1 mM l -glutamine, 0.1 mM β-mercaptoethanol, and 1 mM nonessential amino acids supplemented with 4 ng/ml bFGF on mitotically-inactivated MEF feeder layers and matrigel.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay