Fig. 7.

Bach1 and c-Myc Nuclear Translocation is Dysregulated by Chronic Hyperoxia. MEF cells were cultured either at 5% or 40% oxygen prior to pretreatment. Cells were either not pretreated with H₂O₂ or pretreated with a non-damaging, signaling level (1.0 μM H₂O₂ in a final volume of 2.0 ml in 6 well plates) for 1 h and then allowed to recover for either 1 h or 18 h post treatment. Cytosolic and nuclear fractions were isolated. All treatments were done in replicates of 6 (n = 6). (A) The levels of c-Myc and Bach1 were assessed by Western blot in the cytosolic versus nuclear fractions 1-h and 18-h after initial H₂O₂ pretreatment and normalized either to GAPDH (cytosolic fraction) or LAMIN (nuclear fraction) loading controls. (B) Quantification of c-Myc levels within cytosolic and nuclear fractions. MEF cells cultured at 5% oxygen, showed an increasing accumulation of c-Myc within the nuclear fraction by 18 h post-pretreatment, with no change in the cytosolic fraction. MEF cells cultured at 40% oxygen, showed increased baseline amounts of c-Myc in both nuclear and cytosolic fraction, but no adaptive accumulation in either fraction post-pretreatment. (C) Quantification of Bach1 levels within the cytosolic and nuclear fractions. MEF cells cultured at 5% oxygen, showed an increasing amount of Bach1 at 1 h and 18 h in the cytosolic fraction, whereas, Bach1 accumulated after 18 h post-pretreatment in the nuclear fraction. In contrast, MEF cells propagated at 40% oxygen showed a baseline increase in Bach1 levels in the cytosolic and nuclear fractions, but no difference in cells either not pretreated or pretreated with hydrogen peroxide. All data are expressed as means ± standard errors and Statistically significant differences were indicated by * (p < 0.05), ** (p < 0.01), *** (p < 0.001).