Increases in Retrograde Injury Signaling Complex-Related Transcripts in Central Axons following Injury

Figure 5

Importazole blocks importin -mediated nuclear import in hippocampal cells. (a) Control cells expressing NFAT-GFP were treated with ionomycin to induce nuclear import of NFAT-GFP. NFAT-GFP localizes within nucleus (Hoechst, arrows) and surrounding regions (overlay, arrows). Inset is expanded for more clear visualization. (b) Cells expressing NFAT-GFP were treated with 18 μM importazole prior to treatment with ionomycin, and nuclear localization was compared to controls in the presence of ionomycin (quantified in c). NFAT-GFP is excluded from nucleus (Hoechst, arrows) but retains extranuclear localization (overlay, arrows). Inset is expanded for more clear visualization. (c) Percentage of cells with nuclear NFAT-GFP. 150 or more cells were counted under each condition. Student’s t-test with Bonferroni’s correction . (d–e) Live/dead assay of either (d) control or (e) IPZ treated cells treated with ionomycin after transfection shows no toxicity (red) and high viability (green) in both control and treated cells. (f) Given the possibility that only nonneuronal cells were affected by IPZ, we tested whether cells expressing NFAT-GFP were immunolabeled with the axonal marker (SMI-31, red) after IPZ treatment. SMI-31 positive cells indeed expressed NFAT-GFP, thus indicating that neuronal cells were capable of being affected by IPZ treatment. NFAT-GFP signal is observed in the proximal axons. Arrows indicate absence of GFP signal within the nucleus. SMI-31 also cross-reacts with nuclear intermediate filaments, thus resulting in red-labeled nuclei. GFP images have been contrast-enhanced for visualization purposes. Bar is 50 μm. Values represent mean ± SEM.

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