Fig. 3.

(A) H₂O₂ exposure causes an increase in Pa200 protein levels. MEF cells were pretreated with 0, 1, 10, or 100μM H₂O₂, 24 h later samples were harvested and run on Western blots which were then screened with antibodies against either Pa200 or the loading control β-tubulin. Sample band intensity was measured and adjusted based on β-tubulin band intensity. Values are means ± SE where n = 3. The inset shows a representative blot. (B) By blocking the adaptive increase in Pa200, the H₂O₂ induced increase in oxidative stress tolerance is blunted. MEF cells treated with either Pa200 or (scrambled) siRNA for 24 h to block induction. Samples were then transiently adapted to oxidative stress by pre-treatment with 1μM of H₂O₂, for 1 h. Following a 24 h adaptation period, both adapted and non-adapted cells were challenged by incubation with a high dose of 100μM H₂O₂. Cell counts were then taken using a cell counter 24 h later. Results are means ± S.E. where n = 3. (C) Exposure to a toxic level of H₂O₂ causes the formation of Pa200 foci in the nucleus. MEF cells were exposed to 1.0mM H₂O₂ for 1hr. Immunocytochemistry was then performed and cells were stained with anti-Pa200. (D) Exposure to low, adaptive doses of H₂O₂ also results in the formation of nuclear Pa200 foci. Conditions were identical to those of panel C., except that 0, 1.0μM, or 10.0μM H₂O₂ exposures were used.