Figure 14.

(A) Illustration of the 5′- constructs of Prdx6 promoter (−918/+30 bps) linked to CAT reporter plasmid showing E-Box and ARE site position with wild-type and mutant sequences. (B) Age-related reduction in transcriptional activity of Prdx6 promoter was reactivated by Metformin in hLECs derived from lenses of aging subjects. Histograms showing the Prdx6 promoter activity in absence or presence of Metformin in different ages of hLECs as indicated. Data represents the mean ± S.D. from two independent experiments. Younger age (23 y) vs. aging sample (55 y and 71 y); untreated vs. Metformin-treated; * p < 0.05; ** p < 0.001. (C–E) Transactivation assay with mutant promoters of Prdx6 at E-Box and ARE sites revealed that both Bmal1 and Nrf2 were essential for optimum activation of Prdx6 transcription in SRA-hLECs by Metformin. (C) Transcription activation of the wild-type (WT) and mutant (mut) promoters of Prdx6 revealed that both Nrf2 and Bmal1 were critical elements to activate Prdx6 promoter activity. SRA-hLECs were transfected with WT-pCAT-Prdx6 promoter wild type (WT) plasmid or its mutant promoter plasmids at E-Box (E-Box mut) or ARE (ARE-mut) or at both E-Box and ARE (E-Box-mut + ARE-mut) sites as shown in Figure A. 48 h later, transfectants were treated with vehicle control or Metformin for 24 h as indicated, and Prdx6 transcription activity was measured. All histograms are presented as mean ± S.D. values derived from three independent experiments, ** p < 0.001. (D) Metformin failed to activate Prdx6 transcription in Bmal1-depleted SRA-hLECs. Stably infected SRA-hLECs either with GFP-linked LV control or LV shRNA specific to Bmal1 as described in Materials and Methods and Bmal1- knock down efficiency was confirmed by Western blot analysis (Da). LV shControl or Bmal1-depleted SRA-hLECs were transiently transfected with human Prdx6 promoter (−918/+30) fused with CAT reporter plasmid. 48 h later, the transfectants were treated with Metformin for 24 h as indicated in figure. Cell lysates were prepared and measured the Prdx6 promoter activity. All histograms are presented as mean ± S.D. values derived from three independent experiments, ** p < 0.001. (E) Nrf2-deficiency in SRA-hLECs resulted in significantly reduced Prdx6 transcription in presence of Metformin. SRA-hLECs were transfected with either mock, negative control shRNA or Nrf2 shRNA. Nrf2 knock down in SRA-hLECs was confirmed by Western blot analysis (Ea). The transfectants containing sh-control or sh-Nrf2- SRA-hLECs were transiently transfected with pCAT-Prdx6 promoter (−918/+30) plasmid. 48 h later these transfectants were treated with Metformin for 24 h and promoter activity was assessed. All histograms are presented as mean ± S.D. values derived from three independent experiments, ** p < 0.001.