Fig. 5.

A and B: high glucose augments rDNA transcription, which requires activation of UBF. GECs were transfected with rDNA-IRES-Luc reporter construct alone (A) or cotransfected with UBF S388G inactive mutant or an empty plasmid vector (B) for 24 h; they were then incubated with or without high glucose for an additional 24 h. The cells were harvested at the end of experiment, and lysates were subjected to luciferase activity assay as described in materials and methods. rDNA-IRES-Luc activity measured as arbitrary units/mg protein was expressed as %control. Composite data from 4–6 experiments done in triplicates are shown in histograms. *P < 0.001, by ANOVA (B). Student's t-test was employed to assess changes between 2 groups (A). C and D: Erk and mTORC1 activation is required for high-glucose-stimulated rDNA transcription in GEC cells. GECs were cotransfected with rDNA-IRES-Luc reporter construct and empty vector or inactive mutants of Erk (C) or p70S6 kinase (D) followed by treatment with or without high glucose. Luciferase activity assay was performed as described in A. Composite data from 4 experiments are shown in histograms. *P < 0.001 and #P < 0.05, by ANOVA.