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. Author manuscript; available in PMC: 2014 Apr 1.
Published in final edited form as: Free Radic Biol Med. 2012 Dec 27;57:119–131. doi: 10.1016/j.freeradbiomed.2012.12.014

Fig. 7.

Fig. 7

Antioxidant-mediated stabilization and overexpression of Nrf2 or Bcl-xL reduced etoposide-mediated DNA fragmentation leading to cell survival. (A) Stabilization/overexpression of Nrf2 or Bcl-xL decreased DNA fragmentation. Hepa-1 cells were treated with increasing concentrations of etoposide for 30h followed by treatment with DMSO or tBHQ for additional 24h (upper panel). Similarly Hepa-1 cells were transfected with pcDNA of Flag-Bcl-xL plasnid for 24h and treated with increasing concentrations of etoposide for 30h (middle panel). Control HEK293 and HEK293-Nrf2 cells were treated with increasing concentrations of etoposide for 24h followed by treatment with 2µg/ml of tetracycline for 24 h (lower panel). The cytoplasmic histone-associated DNA fragments [mono and oligonucleosomes) were quantified using Cell Death Detection ELISA kit (Roche) and plotted]. The experiment was repeated thrice. Each point represents a mean ± SD and normalized to the value of the corresponding control cells.(B) Nrf2 or Bcl-xL knockdown increased DNA fragmentation and decreased cell survival. Hepa-1 cells were transfected with control siRNA 100 nM or two different concentration of Nrf2 siRNA (50 nM and 100 nM) (upper panel) or Bcl-xL siRNA (lower panel). After 24 hours of transfection, eighty micrograms of cell extracts were immunoblotted with anti-Nrf2, Bcl-xL and anti β-actin antibodies.(C)Hepa-1 cells were plated at a density of 5000 cells per well in 24 well plates and cells were transfected with control siRNA 100 nM or two different concentration of Nrf2 siRNA and Bcl-xL siRNA (50 nM and 100 nM) separately for 24h. This was followed by treatment with etoposide (20 µM) for additional 30 h and etoposide mediated histone associated DNA fragmentation was analysed (upper panel). (D) Cell survival assay. Hepa-1 cells were plated at a density of 5000 cells per well in 24 well plates, and transfected with Nrf2 or Bcl-xL siRNA and treated with DMSO or etoposide 30h as indicated. Cells were incubated with fresh MTT solution for 2h at 37°C and absorbance at 570 nm was measured. The experiment was repeated thrice. Each point represents a mean ± SD and normalized to the value of the corresponding control cells.