FIGURE 3.
Prx5 helps to maintain ROS homeostasis in MEFs. A, prx1-/- MEFs were transduced with a retrovirus expressing a Prx5 shRNA or a control retroviral vector. Stable, pooled, puromycin-resistant clones were then analyzed for Prx5 expression by immunoblotting. β-Tubulin was used as a loading control. Note that the shRNA reduced Prx5 levels to those seen in prx1+/+ cells. B, MEFs from A were stained with 0.25 μm CM-H2-DCFDA and analyzed by flow cytometry. Representative results from at least three experiments are depicted here. Strains are as follows. a, prx1+/+; b, prx1-/-; c, prx1-/- + prx5 shRNA; d, prx1-/- + control shRNA. The inset shows quantification of the histograms as previously described. Note the approximate 2-fold increase in ROS in prx1-/- + prx5 shRNA cells when compared with prx1-/- cells. *, p value was significant when comparing prx1-/- cells expressing control shRNA with those expressing prx5 shRNA (p = 0.03). C, prx1-/- cells expressing either the prx5 shRNA or control shRNA were stained with CM-H2-DCFDA and then imaged by fluorescence microscopy, as described previously. Note that the prx5 shRNA-expressing cells demonstrate a more intense nuclear and cytoplasmic accumulation of ROS than the control shRNA-expressing cells.