Fig. 2.

Cell Growth and Proteasomal Chymotrypsin-like Activity. (A) Growth rate is attenuated with increasing O₂ culturing concentration. Cells were seeded at 1 × 10⁵ in 6-well plates at either 5%, 21%, or 40% O₂, with 3 replicates (n = 3) per oxygen concentration. After 3 days of incubation, cells were washed twice with PBS, detached with trypsin, and counted with a hemocytometer. (B) Pretreatment with 1.0 μM H₂O₂ (per 500,000 cells) results in a protective effect against a subsequent challenge dose of 3.0 mM H₂O₂ per 500,000 cells, in MEFs cultured at 5% O₂ but not in MEFs cultured at 40% O₂. Cells were either cultured at control conditions of 5% oxygen (5%), 40% oxygen for 2 weeks (40%), or cultured at 40% oxygen and then transferred back to 5% for 2 weeks to de-adapt the cell lines before assays. Cells were pre-exposed to 1.0 μM H₂O₂ (per 500,000 cells) 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 18 h before the challenge dose. The challenge dose was administered for 1 h and cells were allowed to recover for 24 h before cell counts were taken. (C) A signaling treatment of 1.0 μM H₂O₂ (per 500,000 cells) increases proteolytic capacity in MEF's cultured at 5% O₂ but fails to increase proteolytic capacity in MEFs cultured at higher O₂ concentrations. An oxidative signaling dose of 1.0 μM H₂O₂ was administered for 1 h and cells were allowed to recover for 18 h. (D) De-adaptation to hyperoxia allows restoration of H₂O₂ adaptive responses. Cells cultured at 40% O₂ for 28 days were transferred back to 5% O₂ for 21 days. Cells were either cultured in standard media or media with 1 μM H₂O₂ (final concentration) for 1 h and then harvested 18 h after. Chymotrypsin-like activity was measured. Data are expressed as means ± standard errors and statistically significant differences are indicated as * (p < 0.05), ** (p < 0.01), *** (p < 0.001).