Journal:

Article Title: Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

doi: 10.1016/j.bbrc.2010.04.028

Figure Lengend Snippet: Luciferase assay results under following conditions: (A) pGAMPAC-ChREBP, pG5-luc and pRL-TK, (B) pGAMPAC-CA-ChREBP, pG5-luc and pRL-TK, (C) pGAMPAC-ChREBP, pG5-luc, pRL-TK and indicated amount of pcDNA3.1-cG6Pase or pcDNA3.1 were cotransfected into 832/13 cells. Transfected cells were incubated in media with low glucose concentration (2.5 mM) overnight, and then treated with 2.5 (open bar) or 27.5 (solid bar) mM glucose, and for (A) and (B), also indicated concentrations of D-mannoheptulose, for 6 hrs. * p< 0.05; ** p<0.005 vs vehicle treated at 27.5 mM glucose (A) or empty vector transfected at 27.5 mM glucose (C).

Article Snippet: UAS-driven luciferase reporter pG5-luc and the internal control TK promoter driven Renilla luciferase expression plasmid pRL-TK were from Promega (Madison, WI).

Techniques: Luciferase, Transfection, Incubation, Concentration Assay, Plasmid Preparation

Journal:

Article Title: Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

doi: 10.1016/j.bbrc.2010.04.028

Figure Lengend Snippet: (A) Over-expression of GCK in 832/13 changed kinetic behavior of GAL4-ChREBP. 832/13 cells were infected with retrovirus which over-expresses GCK as described in Materials and Methods. Infected cells or uninfected control cells were transfected with pGAMPAC-ChREBP, pG5-R2.2luc and pRL-TK, and treated with low glucose over night, and then treated with indicated concentrations of glucose for 3 hrs before luciferase assay. (B) 832/13 cells overexpressing GCK and the control cells were cultured under low glucose (2.5 mM) over night and then treated with 0, 20 and 30 mM glucose for 1 hr followed by G-6-P measurement. (C) pGAMPAC-ChREBP, pG5-luc, pRL-TK and indicated amount of pCMX-GCK were transfected into HEK293T cells. The total amount of transfected DNA was held the same by adding appropriate amount of vector DNA. The transfected cells were then treated with low (5.5 mM) or high (27.5 mM) glucose concentration for 24 hrs before luciferase assay. * p < 0.05; ** p<0.005 vs vector transfected control cells at the same glucose concentration or as indicated (C).

Article Snippet: UAS-driven luciferase reporter pG5-luc and the internal control TK promoter driven Renilla luciferase expression plasmid pRL-TK were from Promega (Madison, WI).

Techniques: Over Expression, Infection, Transfection, Luciferase, Cell Culture, Plasmid Preparation, Concentration Assay

Journal:

Article Title: Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

doi: 10.1016/j.bbrc.2010.04.028

Figure Lengend Snippet: (A) pGAMPAC-ChREBP, pG5-luc, pRL-TK and indicated amount of pCMX-G6PD were infected into 832/13 cells. The total amount of transfected DNA was held the same by adding appropriate amount of vector DNA. Transfected cells were subsequently cultured under 2.5 mM low glucose over night, and then treated with either low (2.5 mM) or high (27.5 mM) glucose for 6 hrs before luciferase assay. * p< 0.05; ** p< 0.005 vs vector only transfected at 27.5 mM glucose. (B) Real-time PCR measurement of mRNA content of G6PD in 832/13 cells infected with lentivirus expressing shRNA against G6PD (knockdown) or its scrambled counterpart (ctrl). Data are normalized to mRNA level of the Eukaryotic Elongation factor 1 gamma (EEF1g). * < 0.05 vs ctrl. (C) Luciferase assay: pGAMPAC-ChREBP, pG5-luc and pRL-TK were transfected into 832/13 cells infected with lentivirus expressing either shRNA against G6PD (knockdown) or scrambled shRNA (ctrl). The transfected cells were then cultured in low glucose overnight, and treated with either 2.5 mM or 27.5 mM glucose for 6 hrs before luciferase assay. * < 0.05 vs ctrl at 27.5 mM glucose.

Article Snippet: UAS-driven luciferase reporter pG5-luc and the internal control TK promoter driven Renilla luciferase expression plasmid pRL-TK were from Promega (Madison, WI).

Techniques: Infection, Transfection, Plasmid Preparation, Cell Culture, Luciferase, Real-time Polymerase Chain Reaction, Expressing, shRNA

Journal:

Article Title: Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

doi: 10.1016/j.bbrc.2010.04.028

Figure Lengend Snippet: (A) pGAMPAC-ChREBP, pG5-luc, and pRL-TK were transfected into 832/13 cells, which were subsequently cultured under low glucose overnight, and then treated with indicated concentrations of 2-DG and/or D-mannoheptulose for 6 hrs before luciferase assay. DG15, 15 mM 2-DG; DG30, 30 mM 2-DG; M10, 10 mM D-mannoheptulose:* p < 0.05; ** p < 0.005 vs ctrl; # p< 0.05 vs DG30. (B) pGAMPAC-ChREBP, pG5-luc, and pRL-TK were cotransfected with control vector, pCMX-PFK1 and/or pIRESpuro-iPFK2 (0.15 μg each) into 832/13 cells. The total amount of transfected DNA was held the same by adding appropriate amount of vector DNA. Transfected cells were subsequently cultured under low glucose overnight, and then treated with either 2.5 mM or 27.5 mM glucose for 6 hrs before luciferase assay. ** p < 0.005 vs ctrl at high glucose.

Article Snippet: UAS-driven luciferase reporter pG5-luc and the internal control TK promoter driven Renilla luciferase expression plasmid pRL-TK were from Promega (Madison, WI).

Techniques: Transfection, Cell Culture, Luciferase, Plasmid Preparation