Fig. 5.

Increasing ROS with H₂O₂causes cytochrome c release independent of Bax.A, Effect of different concentrations of H₂O₂ on ROS levels (CM-H₂DCFDA intensity) in NGF-maintained neurons from the three baxgenotypes and on ROS levels inbax^−/− neurons deprived of NGF for 18 hr. After cells were loaded with CM-H₂DCFDA, the dye was washed out, and cultures were exposed to H₂O₂ in the incubator for 20 min. They were then washed three times with L-15 medium and left in the second wash for confocal microscopy. The line shows a linear regression of all the data (R > 0.99).n = 45–128 neurons. B, Cytochromec was lost frombax^−/− neurons after H₂O₂ exposure. Top panels, Paired DIC and fluorescent micrographs of a control (+NGF)bax^−/− cell immunostained for cytochrome c. Bottom panels, Similar cells after treatment with H₂O₂ (2 mm). Cultures for were exposed to H₂O₂ in NGF-containing medium for 30 min. They were then washed twice with NGF-containing medium without H₂O₂ and returned to the incubator for 30 min before processing for immunocytochemistry. Western blots of total cytochrome c (mitochondrial and cytoplasmic) and tubulin in cultures exposed to H₂O₂ confirmed loss of cytochrome c without tubulin loss (data not shown).C, Effect of different concentrations of H₂O₂ on cytochrome c release in NGF-maintained neurons of the three bax genotypes and onbax^−/− neurons deprived of NGF for 18 hr. Cells were exposed to the indicated H₂O₂concentrations for 30 min, washed two times with culture medium containing or lacking NGF, as appropriate, and returned to the incubator for another 30 min to assure degradation of released cytochrome c (Deshmukh et al., 1998; Neame et al., 1998; Kirkland and Franklin, 2001). Release was determined as in Figure 2. Symbols have the same meaning as inA. Data are from 3–11 cultures. D, Time-dependent effect of H₂O₂ on cytochromec release. Concentrations of H₂O₂ that had little effect on cytochromec redistribution after 30 min of exposure caused release when neurons (bax^+/+) were treated with these concentrations for longer periods. H₂O₂ was replenished every 30 min. Data are from three to five cultures. The cytochrome c status of 50–100 neurons was determined for each culture in C andD.