Journal: The Journal of Biological Chemistry
Article Title: Hexokinase 2 and nuclear factor erythroid 2–related factor 2 transcriptionally coactivate xanthine oxidoreductase expression in stressed glioma cells
Figure Lengend Snippet: SIRT6 affects cell viability through regulation of redox homeostasis. A , Western blot demonstrating SIRT6 levels in IL-1β–treated glioma cells in the presence or absence of glucose. Blots are representative images of three independent experiments showing similar results. Blots were reprobed for β-actin to establish equivalent loading. Densitometry data depicting -fold change in SIRT6 expression over control under the indicated treatment conditions normalized to corresponding loading controls are shown. siRNA-mediated knockdown of SIRT6 increases ( B ) and SIRT6 overexpression decreases ( C ) viability of glioma cells deprived of glucose and treated with IL-1β as determined by MTS assay. Insets in B and C show knockdown efficiency of SIRT6 siRNA and increased SIRT6 expression upon transfection with SIRT6 overexpression construct. D and E , DHE fluorescence intensity showing that ROS generation in glucose-deprived IL-1β–treated cells is SIRT6-dependent. F , SIRT6 regulates SOD2 expression under a combination of inflammatory and metabolic stresses. Densitometry data depict -fold change in SOD2 expression over control under the indicated treatment conditions normalized to corresponding loading controls. Each data point in the scatter plots represents average absorbance values depicting glioma cell viability ( n = 4) ( B and C ) and fluorescence values depicting ROS levels under the indicated treatment conditions from independent experiments ( n = 3) ( D and E ). − G denotes glucose-free DMEM. NSsiRNA , nonspecific siRNA; SIRT6OE , SIRT6 overexpression. Two-tailed paired Student's t test ( B and C ) and one-way ANOVA (Bonferroni's multiple comparison test) ( A , D , E , and F ) were used for statistical analysis. Error bars represent S.E. *, p
Article Snippet: Transfection with 70 n m duplex HK2, 50 n m SIRT6, and Nrf2 or nonspecific siRNA (Thermo Fischer Scientific) was carried out using Lipofectamine RNAiMAX reagent (Life Technologies-Invitrogen) as described previously ( ).
Techniques: Western Blot, Expressing, Over Expression, MTS Assay, Transfection, Construct, Fluorescence, Two Tailed Test