Figure 3.
SRF protects cortical neurons from trophic withdrawal. A, Representative photomicrographs of cortical neurons. Primary cortical neurons were transfected with vector control or an expression vector for wt SRF (4 μg). In all cases, a β-galactosidase expression vector was A, Representative photomicrographs of cortical neurons. Primary cortical neurons (-S), serum-containing conditioned medium (+S), or 10 ng/ml BDNF as indicated. Twenty-four hours later, cells were fixed and immunostained with an antibody against β-galactosidase to identify transfected cells (top panels). Hoechst 33342 staining was used to visualize nuclear morphology (bottom panels). Arrows point to the nuclei of transfected cells. The arrow-pointed nuclei in d are fragmented and condensed, characteristic of apoptosis, whereas those in panels b, f, and h are evenly stained, indicating healthy cells. B, Cells expressing SRF maintain viability after serum withdrawal. Cortical neurons were transfected with vector control or wt SRF (4 μg) and subjected to serum deprivation (-S) for 24 hr as in A. The cells were then incubated with 10 nm Mitotracker Red CMXRos to stain mitochondria (middle panels). Thirty minutes later, the cells were fixed and stained with an antibody against β-galactosidase to identify transfected cells (top panels) and with Hoechst dye 33342 to visualize nuclear morphology (bottom panels). Note that the cell transfected with the wt SRF has evenly stained nuclei and exhibits a punctate staining pattern for the mitotracker (arrow), indicating that it is nonapoptotic and viable. In contrast, the control transfected cell has typical apoptotic nuclear morphology and diffuse mitotracker staining, indicating that it is apoptotic and nonviable. C, D, SRF protects cortical neurons from trophic withdrawal in a dose dependent manner. Cortical neurons were transfected at DIV 3 with varying concentrations of a wt SRF DNA (0-4 μg). Cells were also cotransfected with a plasmid DNA encoding β-galactosidase as a marker for transfection. The CGN vector DNA was used as a supplement so that all plates have an equal amount of DNA. Two days after transfection, cells were washed twice with serum-free medium and then placed in serum-free medium (-S) or serum-containing conditioned medium (+S). Apoptosis in the transfected cell population (β-galactosidase-stained neurons; C) and the general cell population (D) was scored 24 hr later. Results are averages of five independent experiments ± SEM; ***p ≤ 0.001(ANOVA). NS, Not statistically significant. E, Western analysis for transfected SRF. Cortical neurons were transfected and treated as in C and D. Cell lysates were prepared 2 d after transfection, and 25 μg of total protein was used for Western blotting against SRF. Western analysis of β-actin was used as a loading control. The transfected SRF is expressed in a dose-dependent manner.