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. Author manuscript; available in PMC: 2010 Feb 10.
Published in final edited form as: Cell Stem Cell. 2008 Mar 6;2(3):241. doi: 10.1016/j.stem.2008.01.002

Figure 1. 2-ME withdrawal induces intracellular ROS increase by SIRT1-mediated inhibition of the capacity of p53 to enhance expression of antioxidants.

Figure 1

(A) Changes of intracellular ROS levels in wild-type and SIRT1−/− mES cells, 24 h after 2-ME withdrawal. ROS levels are expressed as the mean ± SEM of intensity of cell fluorescence. Intracellular ROS was measured by monitoring conversion of DCFH-DA to DCF using FACscan. Expression levels of SIRT1 in wild type and SIRT1−/− mES cells are shown in the insert. *, p<0.005 versus 0 h after 2-ME withdrawal. (B) Histograms for intracellular ROS level in wild-type, SIRT1−/−, and SIRT1−/− mES cells infected with control or SIRT1 expressing lentivirus at 0 and 24 h after 2-ME withdrawal. Expression levels of SIRT1 in SIRT1−/− mES cells infected with control or SIRT1 expressing lentivirus are shown in the insert of right lower panel. (C) Changes of mRNA expression levels of GPX1, SESN1 and SESN2 in wild type and SIRT1−/− mES cells 24 h after 2-ME withdrawal. Expression levels were detected by quantitative real time PCR. Fold changes were calculated from β-actin normalized Ct (threshold cycle) values; error bars represent SD of triplicate experiments. (D) The effect of tiron, an antioxidant, on 2-ME withdrawal induced expression of GPX1, SESN1 and SESN2. SIRT1−/− mES cells were incubated with or without 2-ME in the presence of the indicated dose of Tiron (07#x2013;2 mM). Twelve hours later, mRNA expression levels of GPX1, SESN1 and SESN2 were measured by quantitative real time PCR. Fold changes were calculated from β-actin normalized Ct values; error bars represent SD of triplicate experiments. (E) The expression levels of p53 in scrambled shRNA and p53 shRNA expressing wild and SIRT1−/− mES cells, as detected by western blot. Wild-type and SIRT−/− mES cells were infected with control or p53 shRNA expressing lentiviral particles and selected with 2 μg/ml puromycin. (F) Changes of intracellular ROS levels in scrambled shRNA and p53 shRNA expressing wild-type and SIRT1−/− mES cells 24 h after 2-ME withdrawal. ROS levels are expressed as mean ± SEM of intensity of cell fluorescence. (G) Changes of mRNA expression of GPX1, SESN1 and SESN2 in scrambled shRNA and p53 shRNA expressing SIRT1−/− mES cells, 24 h after 2-ME withdrawal. Expression levels were detected by quantitative real time PCR. Fold changes were calculated from β-actin normalized Ct values; error bars represent SD of triplicate experiments.