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Figure 8


Fig. 8. Cdk5-mediated p53 transactivation induces mitochondria-mediated neuronal apoptosis in response to cellular stress. (A) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, and then treated with 5 µM mitomycin C for 9 hours. Total RNA was isolated, and quantitative real-time RT-PCR was performed. The presented results are from three independent experiments and are given as the mean ± s.d. Each experiment was performed in triplicate. *P<0.005. (B) SH-SY5Y cells were pretreated with 10 µM Cdk2/5 inhibitor or an equal volume of vehicle for 30 minutes and then incubated with 5 µM mitomycin C for 12 hours. Cells were triple-labeled with Hoechst (blue), anti-cytochrome C (red) and MitoTracker (green) and then analyzed by fluorescence microscopy. (C) SH-SY5Y cells were transiently transfected with vectors encoding EGFP-Cdk5(D144N) or EGFP, treated with 5 µM mitomycin C or 2 mM SNP for 12 hours and fixed and stained with antibodies against cleaved caspase-3. (i) Representative image of EGFP-Cdk5(D144N)-positive cells. (ii) Percentage of EGFP- or EGFP-Cdk5(D144N)-expressing cells positive for cleaved caspase-3. The data represent the mean ± s.d. from three independent experiments. *P<0.0001. (D) SH-SY5Y cells were transiently transfected with mock vector or vectors encoding Cdk5(D144N), and then treated with 5 µM mitomycin C or 2 mM SNP for 12 hours. Cell lysates were subjected to immunoblotting with the indicated antibodies. (E) SH-SY5Y cells were transiently transfected with mock vector or vectors encoding Cdk5(D144N), and then treated with 5 µM mitomycin C, 2 mM SNP or an equal volume of vehicle. Cell viability was measured 24 hours later by MTT assay. The data represent the mean ± s.d. from quadruplicate determinations. Similar results were obtained in three independent experiments. *P<0.0001. (F) Proposed model for Cdk5-mediated regulation of p53 and subsequent mitochondria-mediated apoptosis.





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