Fig. 7. Sensitivity to oxidative stress in the MIRTD-deficient cells. (A) Maxizyme-expressing cells (Z5, Z7 and Z21) and control cells (L1 and R2) were exposed to medium containing 0.2, 0.4 or 0.6 mM H₂O₂. After 24 h, the cells were stained with Hoechst 33342 (blue, dead and living cells) and propidium iodide (pink, dead cells). Cell viability was obtained by counting 200 cells in each clone exposed to various concentrations of H₂O₂. (B) Z5, Z21, L1 and R2 cells were exposed to 0.4 mM H₂O₂ for the periods indicated. Cell viability was calculated as above. (C) The transfectants (L1, R2, Z7 and Z21) were induced to differentiate into putative neurons with 10 µM retinotic acid in the presence of 1% FBS for 5 days and then exposed to various concentration of H₂O₂ for 12 h in the presence of 1% FBS. Cell viability was assessed as described above. (D) Total protein was prepared from the L1 and Z21 cells exposed to 0.2 mM H₂O₂ at 0, 6, 12 and 24 h. Protein (50 µg) from each sample was subjected to SDS–PAGE and western blotting with antibody against the C-terminal region of the DLST protein. Arrowheads indicate MIRTD and the full-length DLST protein. (E) Poly(A)⁺ RNA was isolated from L1 and Z21 cells exposed to 0.2 mM H₂O₂ at 0, 3, 6, 12 or 24 h. The amounts of MIRTD mRNA were determined by real-time quantitative RT–PCR and normalized to those of DLST mRNA. (F) The amounts of the MIRTD protein were quantified by western blotting of (D) and normalized to those of DLST. (G) Z5, Z21, L1 and R2 cells were exposed to the medium containing menadione at the indicated concentration. After 24 h, cell viability was measured as described in (A). (H) Z21 and L1 cells were incubated in the presence or absence of 10 µM menadione for 3 h and then treated with CM-H₂DCFDA (Molecular Probes) (5 µM) for 1 h. The cells were trypsinized and subjected to flow cytometry to measure the fluorescent signal of DCF with an EPICS ELITE flow cytometer (Beckman Coulter). Twenty thousand cells were measured in each experiment and mean values were obtained. Results in (A–C) and (E–H) are shown as the average ± SD obtained from three or four experiments.