Figure 5

Endogenous production of NO by eNOS overexpression maintains mESC self-renewal after LIF withdrawal. (a) D3-pOct4-eGFP-eNOS mESCs from passage 10 after sorting were cultured for 7 days in the presence (left panel) or absence (medium panel) of LIF or in the absence of LIF plus 400 μM L-NMMA (right panel). GFP intensity was measured by flow cytometry. Cells with fluorescence intensity similar or higher than that observed for mESCs transfected with the pOct4-eGFP construct and cultured for 7 days in the presence of LIF were considered positive for eGFP (M1 population). Each graph is representative of five independent experiments (n=5). (b) qRT-PCR analysis of Oct4, Sox-2 and Nanog in D3-pOct4-eGFP-eNOS mESCs cultured for 7 days under the indicated conditions. β-actin was used as the endogenous control and Ct values were normalized with respect to the Ct of cells cultured in the presence of LIF. Data are the mean ± S.E.M. of three experiments. ^*P≤0.005 versus. cells cultured in the absence of LIF. (c) Western blot analysis of Oct4, Sox-2 and Nanog in D3-pOct4-eGFP mESCs cultured for 7 days under the indicated conditions. Representative images and densitometry quantification are from five independent experiments (d) qRT-PCR analysis of Brachyury expression in D3-pOct4-eGFP-eNOS mESCs. β-actin was used as the endogenous control and Ct values were normalized with respect to the Ct of cells cultured in the absence of LIF. Data are the mean ± S.E.M. of three experiments. ^*P≤0.005 versus cells cultured in the presence of LIF. (e) Immunofluorescence images of cells positive for SSEA-1 in D3-pOct4-eGFP-eNOS mESCs cultured for 7 days in the presence (left panel) or absence (medium panel) of LIF or in the absence of LIF plus 400 μM L-NMMA (right panel). Nuclei were counter-stained with DAPI. Images are representative of three independent experiments. Scale bars are 20 μm