Figure 6

Removal of H₂O₂ rescues Txnrd1^−/− MEFs grown in sparse cell cultures. (a) Cells were seeded at a density of 1000 cells/cm² with supplementation of the indicated amounts of conditioned medium (CM) from high-density cultures, or with catalase, and were cultured for either 24 or 96 h as indicated. Cell proliferation was then estimated by determination of total cellular nucleic acids (n=4–9, ±S.E.M.). Significant differences between the 96 h of Txnrd1^−/− and the other 96 h samples are indicated (*P<0.05; **P<0.01; ***P<0.001; n.s., not significant, P>0.05), and significant differences between the 24 h and 96 h of each treatment are also indicated (^#P<0.05; ^##P<0.01; ^###P<0.001; n.s., not significant, P>0.05). (b) A standard curve with pure catalase was incubated with 10 μM H₂O₂ (37 °C, 10 min), and the fluorescence signal indicating the remaining amount of H₂O₂ was determined using Amplex Red. The equation of the standard curve with R² value is also shown. The corresponding catalase activities of 20% fresh medium (FM) or conditioned medium (CM) are also plotted onto the curve, as shown. According to this titration, 20% CM contained 0.76±0.12 units/ml catalase activity and 20% FM had no catalase activity (n=3–9, ±S.E.M.). (c) A total of 1.5 × 10⁴ Txnrd1^−/− cells, with or without supplementation with catalase (100 units/ml), or Txnrd1^fl/fl, Txnrd1^498Sec and Txnrd1^U498C cells as indicated were seeded onto 96-well microtiter plates in HBSS buffer for 18 h, whereupon extracellular H₂O₂ was measured using Amplex Red (n=4–8, ±S.E.M.). Significant differences are indicated (*compared with Txnrd1^fl/fl MEFs, **P<0.01; ***P<0.001; ^#compared with Txnrd1^−/− MEFs, ^##P<0.01; ^###P<0.001)