Effect of SIRT1 activity on ROS production and FoxO3a transcription activity during CK2 downregulation-mediated senescence. HCT116 and MCF-7 cells were treated with CK2α siRNA (A, C) or pcDNA3.1-HA-CK2α (B, D) in the absence or presence of pECE-flag-SIRT1 (A, C) or pECE-flag-SIRT1 mutant (H363Y) (B, D) for 48 h. (A, B) The cells were incubated with 5 μM CM-H2DCFDA (DCF) or 1 μM dihydroethidium (DHE), as previously described (). Fluorescence intensity was determined by flow cytometry analysis. Representative data from three independent experiments are shown (left panels). The graphs show the relative amount of DCF or DHE fluorescence (right panels). (C, D) Total RNA was extracted from the cells and then RT-PCR was performed using specific primers for CK2α, Cu/ZnSOD, MnSOD, catalase, thioredoxin-2 (Trx2), and peroxiredoxin-5 (Prx5). β-actin was used as a loading control. The graphs show the relative mRNA levels of these antioxidant genes.
