Oxidative stress regulates FoxO1 expression through its self-regulation.

<p>(A) MGCs are treated with 20 μM SP600125 for 12 h and then exposed to 100 μM H₂O₂ for 24 h. The mRNA expression of FoxO1 is detected in MGCs and normalized by the expression level of Actb. (B) FoxO1 protein level in MGCs after treatment with 20 μM SP600125 for 12 h and 100 μM H₂O₂ for 24 h. Actb serves as the internal control. (C) Quantification of relative FoxO1 protein level. The relative protein level of FoxO1 is normalized by the expression level of Actb. (D) MGCs are transfected with pGL3-FoxO1 (W) or pGL3-FoxO1 (M) for 36 h, treated with SP600125 for 12 h, and exposed to 100 μM H₂O₂ for 24 h. Activities are normalized using those of pRL-TK. All results above represent mean ± SEM (n = 3). * <i>p</i> < 0.05.</p

JNK mediated H₂O₂-induced apoptosis in MGCs.

<p>(A) JNK activation is detected after treatment with 20 μM SP600125 for 12 h and 100 μM H₂O₂ for 24 h. (B) MGCs are treated with 20 μM SP600125 for 12 h and then exposed to 100 μM H₂O₂ for 24 h. Cell apoptosis is analyzed by TUNEL assay. Apoptotic nuclei and total nuclei show green and blue fluorescence, respectively. Bar, 20 μm. (C) Quantification of the results shown in Fig 1B. All experiments are performed in triplicate. All results above represent mean ± SEM (n = 3). * <i>p</i> < 0.05; * * <i>p</i> < 0.01.</p

JNK affects the interaction between FoxO1 and 14-3-3 proteins in response to oxidative stress.

<p>MGCs are treated with 20 μM SP600125 for 12 h and then exposed to 100 μM H₂O₂ for 24 h, following precipitation with FoxO1 antibody. Precipitates are loaded for SDS/PAGE analysis and transferred for western blotting with FoxO1 and 14-3-3 proteins antibody.</p

Oxidative stress promotes FoxO1 nuclear localization by mediation of the JNK activity.

<p>(A) MGCs are treated with 20 μM SP600125 for 12 h and then exposed to 100 μM H₂O₂ for 24 h. The expression of FoxO1 protein in cell cytoplasm and nucleus is examined using western blot. Internal controls are TBP in the nucleus and Actb in the cytoplasm. (B) Quantification of relative FoxO1 protein level. The level of expression of Actb or TBP is used to normalize the relative expression level of FoxO1 protein. (C) MGCs transfected with pcDNA 3.1 or FoxO1 expression vectors are treated with 20 μM SP600125 for 12 h and then exposed to 100 μM H₂O₂ for 24 h. Immunofluorescence subcellular localization of FoxO1 is identified using anti-FoxO1 (green), and the nuclei are counterstained with DAPI (blue). Bar, 20 μm. (D) The statistical analysis of FoxO1 in the nucleus and in the cytoplasm. All results above represent mean ± SEM (n = 3). * <i>p</i> < 0.05; * * <i>p</i> < 0.01.</p

KO^<i>t</i>Bu-Promoted C4 Selective Coupling Reaction of Phenols and [60]Fullerene: One-Pot Synthesis of 4‑[60]Fullerephenols under Transition-Metal-Free Conditions

A KO^<i>t</i>Bu-promoted direct coupling reaction of phenols and [60]­fullerene was disclosed. The reaction occurs exclusively at the C4-position of phenols with high regioselectivity and provides an efficient and inexpensive manner to various 4-[60]­fullerephenols in good yields. The electrochemical properties of the products render the method attractive and valuable