Research Paper Volume 10, Issue 9 pp 2284—2315

Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

Figure 12. Enhanced Sp1 binding to Prdx6 promoter in cells transduced with Sumoylation–deficient Prdx6 against oxidative stress. (A and B) SRA-hLECs were transduced with TAT-HA-Prdx6WT and its mutant TAT-HA-Prdx6K122/142R mutated at Sumoylation sites recombinant protein followed by different concentrations of H2O2 (A) or UVB (B) exposure as indicated. ChIP assay was carried out using ChIP grade anti-Sp1 antibody. The DNA fragments were used as templates for qPCR by using primer designed to amplify -342 to +30 region of the human Prdx6 promoter bearing GC-box (Sp1 sites). Histogram shows the amplified DNA with real-time PCR analysis; open bars vs gray bars vs black bars. The data represent mean ± SD from two independent experiments (**p<0.05; *p<0.001). (C) The H2O2 and/or UVB treatment schedule. (D) In vivo DNA binding assay revealed that transduction of Prdx6 and its mutant at K122/142R linked to TAT reactivated binding activity of Sp1 in aging primary hLECs. Primary hLECs of variable ages were transduced with TAT-HA-Prdx6 WT or its mutant TAT-HA-Prdx6K122/142R. ChIP experimentation was conducted using anti-Sp1 antibody. Immunoprecipitated DNA fragments were purified and processed for qPCR analysis using specific primers. Histograms represent the TAT-HA-Prdx6 WT and its mutant-induced enrichment of Sp1 at GC-box (Sp1 binding sites) in Prdx6 gene promoter. Open vs gray and black bars, and gray vs black bar; **p<0.05, *p < 0.001. Data revealed a significant augmentation of Sp1 binding by TAT-HA-Prdx6K122/142R in all ages of LECs, but younger cells were more responsive.