Figure 3.

Roscovitine induced apoptosis of cortical neurons is rescued by inhibiting MEK1. E18 rat embryonic cortical neurons were cultured for 7 d in B27/neurobasal medium and then treated with 20 μM roscovitine, 50 μM PD98095, or both for 2 h and fixed for ICC assay, or lysed for Western assay. (A) Fixed cells were stained for Cdk5 and TUNEL. Most fragmented apoptotic nuclei seen after roscovitine treatment alone (j and l), whereas apoptosis was decreased when Erk1/2 activation was inhibited by PD98095 (n compared with j). Images were captured using a Zeiss LSM510 laser-scanning confocal microscope and managed using Adobe Photoshop software. Bar, 20 μm. (B) Bar graph shows the percentage of cell apoptosis. TUNEL-positive cell counts were obtained from 10 independent fields with a total of 500 cells. Results were expressed as mean ± SEM from three separate treatment groups. (C) Cleaved caspase-3 assay in neurons. Neuronal cell lysates were subjected to Western blot analysis to measure the levels of the phospho-Erk1/2, total Erk1/2, cleaved caspase-3, one of the apoptosis markers, and tubulin. High levels of phosphorylated Erk1/2 (first panel, lane 4) and apoptotic caspase-3 were expressed (third panel, lane 4), reduced in each case after treatment with the MEK1 inhibitor PD98095 (first and third panels, lane 3). Bar graph shows mean density measurement of the cleaved caspase-3. Results are expressed as mean ± SEM of three separate experiments.