Figure 4. PRDX1 knockdown did not interfere with APE1 ability to repair damaged DNA.

(a) Total nuclear extracts derived from the four indicated HeLa cell lines were assayed for APE1 AP endonuclease activity using a 42-mer double stranded oligonucleotide substrate carrying a centrally located AP site. Cleavage of the substrate produced a 20-mer product that was quantified and plotted against the concentration of the nuclear extracts. The AP endonuclease activity measurement is the average of two experiments. (b) Western blot showing that APE1 knockdown did not alter PRDX1 level or vice versa. Total cells extracts were prepared from the following cells HeLa carrying either the LUC or LMP control vector, shPRDX1, shAPE1, or both shPRDX1 and shAPE1 and probed by Western blot using antibodies against APE1 and PRDX1. Anti-ACTβ antibody was used to monitor equal loading of the total extracts. The data are representative of two independent experiments. (c) Survival of cells treated with increasing concentrations of H₂O₂. The HeLa cell lines carrying the empty vector LMP or Luc or knockdown for either PRDX1, APE1 or both were treated with H₂O₂ for 1 h and scored for survivors after 10 days using the clongenic assay. The data are the average of three independent analyses.