Article Figures & Data
Figures
- Fig. 1.
Survival of DRG neurons treated with the phosphotidylinositol-3 kinase inhibitor LY294002. DIC-5, DIC-10, and DIC-21 DRG neurons were treated with doses of LY294002 ranging from 1 to 50 μm for 72 hr, after which time they were stained with crystal violet and counted. The cell count of each group of wells was normalized to that of wells containing untreated neurons maintained in the presence of NGF, and percent survival was determined. Each value represents the average (± SD) of five wells; ∼5000 DRG neurons were originally plated in each well.
- Fig. 2.
Time course of apoptosis in DIC-10 DRG neurons treated with 25 μm LY294002 in the presence of NGF. Neurons were stained with bis-benzamide at the time points indicated and assessed, by fluorescent microscopy, for the presence of chromatin condensation. The percentage of neurons that showed chromatin condensation was determined. Each value represents the average ± SD of three plates. The number of cells counted for each time point ranged from 488 to 601. The time course of NGF withdrawal-induced chromatin condensation (data taken from Tong et al., 1996) is shown for comparison purposes.
- Fig. 3.
Treatment with elevated [K+] blocks LY294002-induced apoptosis in DIC-5 DRG neurons. Neurons were maintained in either standard (5 mm) or elevated (35 mm) K+ for 24 hr then treated with 25 μm LY294002 or subjected to NGF withdrawal (NGF−). Twenty-four hours later, neurons were stained with bis-benzamide, and the percentage of neurons undergoing chromatin condensation was determined. Each value represents the average ± SD of three plates. The number of cells counted for each time point ranged from 580 to 678.
- Fig. 4.
Induction of apoptosis by treatment with LY294002. DIC-5 and DIC-21 DRG neurons were treated with either 10 or 25 μm doses of LY294002 for either 18 or 36 hr and then assessed for TUNEL-positive staining. Induction of apoptosis by NGF withdrawal was also performed for comparison purposes (NGF−). The percentage of neurons that showed TUNEL-positive staining was determined. Each value represents the average ± SD of three plates. The number of cells counted for each time point ranged from 578 to 720.
- Fig. 5.
Treatment of DRG neurons with LY294002 blocks AKT activation. DIC-5 and DIC-21 neurons were maintained in the presence of NGF (lanes 2 and 6), subjected to NGF withdrawal (lanes 3 and 7), or treated with 5 (lanes 4 and 8) or 10 (lanes 5 and 9) μm LY294002 in the presence of NGF for 18 hr before lysis. Equal amounts of protein were loaded on 15% polyacrylamide gels and separated by SDS-PAGE. Proteins were transferred to PVDF membranes, and then each membrane was Western blotted individually for total AKT (top) or Ser473-phosphorylated AKT (bottom) using the PhosphoPlus Akt antibody kit (New England Biolabs). Controls (C, lane 1) provided with the kit consisted of total cell extracts from National Institutes of Health 3T3 cells grown in the presence (bottom) and absence (top) of PDGF.
- Fig. 6.
Ser-63 phosphorylation of c-JUN in DRG neurons subjected to NGF withdrawal. DIC-5 (A, B,D, E) and DIC-21 (C,F) DRG neurons were maintained in the presence (A–C) or absence (D–F) of NGF for 18 hr after which they were fixed in 4% paraformaldehyde. Immunofluorescent staining for Ser-63-phosphorylated c-JUN was performed, and neurons were examined with the use of fluorescent microscopy. Positive staining for Ser-63 phosphorylation of c-JUN consists of dense, nuclear immunoreactivity, and is seen only inD–F. Ser-63 phosphorylation of c-JUN is not blocked by treatment with high [K+] medium (E).
- Fig. 7.
Ser-63 phosphorylation of c-JUN in DRG neurons subjected to treatment with LY294002 in the presence of NGF. DIC-5 (A) and DIC-21 (B) DRG neurons were treated with 25 μm LY294002 in the presence of NGF for 18 hr then fixed in 4% paraformaldehyde. Immunofluorescent staining for Ser-63 phosphorylated c-JUN was performed as in Figure6.
- Fig. 8.
A, Western blot analysis of BAX and BCL-X expression in developing DRG neurons. Protein was isolated from DIC-5, and DIC-21 was maintained in the presence of NGF. Equal amounts of protein (25 μg/lane) were loaded on 15% polyacrylamide gels and separated by SDS-PAGE. Proteins were transferred to PVDF membranes, which were then immunoblotted for BAX or BCL-X expression. These blots are representative of three independent experiments. B, Western blot analysis of BAX and BCL-X expression in dorsal root ganglia of postnatal rats (50 μg of total protein/lane). Methods as in A.
- Fig. 9.
Developmental regulation of bax andbcl-xL transcription. Polyadenylated RNA was isolated from DIC-5 and DIC-21 DRG neurons maintained in medium containing NGF. Quantitative RT-PCR was performed with use ofgapdh mRNA levels as a separate, internal control. Multiple cDNA dilutions and cycle numbers were used to determine the linear PCR range. Radiolabeled PCR products were separated on 10% polyacrylamide gels and visualized on a PhosphorImager. Shown are representative examples of PCR products obtained. Quantitation of each band was performed using ImageQuant, and the ratios ofbax/bcl-xL mRNA are shown. These results are representative of four independent experiments.
