Research Paper Volume 10, Issue 9 pp 2284—2315

Figure 10. Sp1K16R mutated at Sumo1 binding site provided enhanced cytoprotection against oxidative stress. (A and B) SRA-hLECs were transfected with either pEGFP-Vector, pCl-neo-HA-Sp1, or pCl-neo-HA-Sp1K16R and then exposed to different concentrations of H₂O₂ as indicated. After 8h of H₂O₂ exposure, ROS intensity was quantified with CellROX deep red reagent (A). 24h later viability of cells was analyzed by MTS assay (B) as shown. Histogram values represent mean ± SD of three independent experiments. 0 vs 100 vs 150µM H₂O₂ and pEGFP-Vector vs pCl-neo-HA-Sp1 WT vs pCl-neo-HA-Sp1K16R (**p<0.05; *p<0.001). (C and D) SRA-hLECs were transfected with pEGFP-Sumo1 along with either pCMV-Vector (open bars), pCl-neo-HA-Sp1 (gray bars), or pCl-neo-HA-Sp1K16R (black bars), and then exposed to oxidative stress. ROS intensity (C) and cell viability (D) are presented as histograms. Values represent mean ± SD of three independent experiments. 0 vs 100µM H₂O₂ and pEGFP-Sumo1 vs pEGFP-Sumo1 plus pCl-neo-HA-Sp1 WT vs pEGFP-Sumo1 plus pCl-neo-HA-Sp1K16R (**p<0.05; *p<0.001). Sumoylation-deficient Sp1K16R (black bars) showed significantly higher protection and reduced ROS production, indicating that mutant Sp1K16R was more effective in protecting cells from oxidative stress Sumoylation-mediated insults.