The Journal of Neuroscience, December 7, 2005, ():
Regulation of Dendritic Morphogenesis by Ras–PI3K–Akt–mTOR and Ras–MAPK Signaling Pathways
J. Neurosci. Kumar et al.
25: 11288
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental figure 1. Western blot analysis to confirm the efficacy and specificity of the active mutants in activating the designated signaling pathways in Hela cell line. The upper panel (A) illustrates the immunoblots probed for pAkt, pMAPK and total MAPK. HeLa cells were transfected with 4µg RasL61, -S35, -C40 and CA Akt in 6 well plates. After 2 days, the cells were harvested and probed for pAkt and pMAPK. The blots were striped and re-probed for total MAPK as shown in lower panels. Panel (B) shows the quantification (mean +/- SEM) of the normalized immunoreactivity for pAkt and pMAPK. Statistical difference among pAkt group: *** p < 0.001. Difference among pMAPK group: ### p < 0.001. One way ANOVA (Dunnett).
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Supplemental figure 2. Immunocytochemistry analysis to confirm the efficacy and specificity of the active mutants in activating Akt in hippocampal neurons. Cultured CA1/3 hippocampal neurons were co-transfected with EGFP and (A) Empty vector, (B) RasL61, (C) Ras S35, (D) Ras C40, (E) CA PI3K and (F) CA Akt on 7DIV and fixed for immunostaining on 15DIV. EGFP-transfected neurons (green), and pAkt473-positive neurons (red, Cy3 secondary antibody) are shown in the adjacent panel for the equivalent field. Note the prominent immunostaining of the neurons transfected with RasL61, RasC40, CA PI3K and CA Akt as compared to the other groups. Scale bar: 100µm.
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Supplemental figure 3. Immunocytochemistry analysis to confirm the efficacy and specificity of the active mutants in activating MAPK in hippocampal neurons. Cultured CA1/3 hippocampal neurons were co-transfected with EGFP and (A) Empty vector, (B) RasL61, (C) Ras S35, (D) Ras C40, (E) CA PI3K and (F) CA Akt at 7DIV and fixed for pMAPK immunostaining at 15DIV. The merged DIC and EGFP fluorescent images illustrate the morphology of the transfected cells (green) and the adjacent untransfected cells. The pMAPK immunostaining (red) is illustrated in the adjacent lower panels. The arrows denote the pMAPK immunostaining in the transfected cells. Panel (G) shows the quantification (mean +/- SEM) of the immunoreactivity. *** p< 0.001. One way ANOVA (Dunnett); n>50 cells for each group. Scale bar: 50 µm.
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Supplemental figure 4. Biochemical and microscopic assessment of long-term inhibition of MAPK, PI3K or mTOR on hippocampal neurons. Cultured CA1/3 hippocampal neurons were treated with (A) vehicle (DMSO), (B) 50 µM LY294002, (C) 1 µM rapamycin and (D) 10 µM U0126 on 7DIV, imaged on 15DIV and then harvested for western blot. All groups showed comparable cell density and healthy cell morphology (smooth and bright phase imaging). Note that a decrease in soma and dendrite size in cells treated with LY294002 or rapamycin can be easily seen even under the low power phase images. Panel (E) shows representative western blots for pAkt, pMAPK and pS6. The pMAPK blot was stripped and then re-probed for total MAPK (lower panel). Panel (F) shows the quantification for western blots from 3 independent experiments. Values are mean +/- SEM. Statistical difference for groups are depicted as: *** (p< 0.001, pAkt groups), ### (p< 0.001, pMAPK) and $$$ (p<0.001, pS6 groups). Scale bar: 100µm.
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Supplemental figure 5. Long-term inhibition of PI3K, MEK or mTOR signaling on cell survival. (A) The micrograph panel illustrates the propidium iodide (PI) and TUNEL assay performed on 15DIV CA1/3 neurons treated with LY294002 (50 µM), U0126 (10 µM), LY+U0126 or rapamycin (1 µM) for a period of 8 days starting from 7DIV. The lower panel (B) illustrates the quantification (mean +/- SEM) for the percentage of cells positive for PI or TUNEL immunofluorescence for long-term (8 days) and short-term (24hr) treatment with pharmacological inhibitors. 5 µg/ml PI was added directly to the culture medium and incubated at 370 C for 10min. Cells were washed, fixed with 4% paraformaldehyde and permeabilized with 0.2 % Triton X-100, and processed for TUNEL staining. No significant increase in cell death was observed for treatments with any single inhibitor, while there was a significant increase in both plasma membrane (PI assay) and DNA damage (TUNEL assay) when both the MEK and PI3K signaling pathways were chronically blocked. A total of 600-900 cells was counted from randomly selected DIC images from 2 independent experiments. ** p < 0.01, *** p < 0.001, One way ANOVA (Dunnett). Scale bar: 50 µm.
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Supplemental figure 6. Immunocytochemistry analysis to confirm the efficacy and specificity of the Ras mutants in activating p-mTOR in hippocampal neurons & morphometric analysis of RasL61S35 and Rasl61C40 mutants. RasL61C40 which preferentially activates the PI3K-Akt pathway demonstrated a significant increase in phospho-mTOR (ser 2448) immunofluorescence as compared with control or with RasL61S35 mutant thus verifying that indeed it’s only –C40 mutant which can activate mTOR signaling. The p-mTOR antibody was obtained from Cell Signaling (Cat # 2971S), and used at 1:200 concentration. Cultured CA1/3 hippocampal neurons were co-transfected with EGFP and (A) Empty vector, (B) RasL61S35 and (C) RasL61C40 on 6 DIV and fixed for p-mTOR immunostaining at 15DIV. The EGFP fluorescent images illustrate the morphology of the transfected cells (green) and the p-mTOR immunostaining (red) is illustrated in the adjacent lower panels. The lower Panel (D) shows the quantification (mean +/- SEM) of the immunoreactivity. * p< 0.05 (One way ANOVA, Dunnett). n=33, 42 and 53 cells for control, RasL61S35 and Rasl61C40 group. Scale bar: 20 µm. The data shown is from a single experiment from 2 sister coverslips for each group. Another independent experiment showed comparable and significant result. (E) and (F) depicts the quantification of dendritic caliber and primary order dendrite number respectively. RasL61C40 had significantly thicker caliber and more number of primary order dendrite number. *** p< 0.001 (One way ANOVA, Dunnett).
