Figure 4.

Induced apoptosis of cortical neurons by Cdk5 siRNA is rescued by inhibiting MEK1 activity. E18 rat embryonic cortical neurons were cultured for 5 d in B27/neurobasal medium and then transfected with 1) control siRNA, 2) Cdk5 siRNA, and 3) Cdk5 siRNA plus 50 μM PD98095 treatment. After 24 h of transfection, cells were fixed and/or lysed. (A) Western blots of cell lysates to measure the levels of the phospho-Erk1/2, cleaved caspase-3, and tubulin. Only in the presence of the Cdk5 siRNA were high levels of phosphorylated Erk1/2 and apoptotic caspase-3 expressed (lane 2), these were reduced in each case after treatment with the MEK1 inhibitor PD98095 (lane 3). Lanes 2 and 3 show reduced expression of cdk5 in the presence of Cdk5 siRNA. Lane 1 shows Cdk5 expression in presence of con siRNA. (B) Bar graph shows the percentage of apoptosis in each situation. Caspase-3–positive cell counts were obtained from 10 independent fields with a total of 500 cells. Results are expressed as mean ± SEM from three separate treatment groups. (C) ICC assay of cells after 24 h of transfection shows p-Erk1/2 and caspase-3–positive cells. Increased p-Erk1/2 and apoptotic cells seen only in b and d when Cdk5 activity is reduced by Cdk5 siRNA. Expressions of both were decreased when Erk1/2 activation was inhibited by PD98095 (f compared with b). Images were captured using a Zeiss LSM510 laser-scanning confocal microscope and managed using Adobe Photoshop software. Bar, 20 μm.