Fig. 3.

Hyperoxia Increases 20S Proteasome Subunit Levels but Abrogates their Adaptive Responses to H₂O₂. MEF cells were chronically cultured at 5% or 40% prior to pretreatment. Cells were either pretreated with a non-damaging amount (1.0 μM) of H₂O₂ in a final volume of 2.0 ml in 6 well plates, for 1 h (or used as controls) and then allowed to recover for either 1 h or 18 h post treatment. All treatments were done in replicates of 6 (n = 6). (A) Amounts of the three proteolytic subunits of the 20S proteasome (β1, β2, β5) were assessed by Western blot and normalized to the actin loading control. MEF cells propagated at 5% showed increased amounts of the three subunits, following H₂O₂ pretreatment, whereas the MEF cells propagated at 40% showed increased baseline levels, but no further adaptive increase following H₂O₂ pretreatment. (B) Quantification of the amount of 20S β1 subunit. (C) Quantification of the amount of 20S β2 subunit. (B) Quantification of the amount of 20S β5 subunit. All data are expressed as means ± standard errors. Statistically significant differences in 5% O₂ cultured cell lines to the 5% O₂ control were indicated by * (p < 0.05), ** (p < 0.01), *** (p < 0.001). Statistically significant differences in the 40% O₂ cultured cell line to the 5% O₂ control were indicated by ^# (p < 0.05), ^## (p < 0.01), ^### (p < 0.001).