JCS03468 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A) SH-SY5Y cells were transfected with p53-targeting or scrambled siRNAs, and then treated with 5 μM mitomycin C or 2 mM SNP for 24 hours. Cell viability was measured by MTT assay. Each bar represents the mean ± s.d. from quadruplicate determinations. Similar results were obtained in three independent experiments. *P<0.0001. (B) SH-SY5Y cells were treated as described in A for 12 hours, and cell lysates were subjected to immunoblot analysis with the indicated antibodies. (C) SH-SY5Y cells were transiently transfected with DsRed-p53(R175H) or DsRed, treated with mitomycin C or SNP for 18 hours and then subjected to TUNEL assays. Cells were triple-labeled with Hoechst (blue), DsRed-p53(R175H) (red) and TUNEL (green), and then analyzed by fluorescence microscopy. (i) Representative image of DsRed-p53(R175H)-positive cells. (ii) Percentage of DsRed- or DsRed-p53(R175H)-expressing cells that were TUNEL-positive. The data represent the mean ± s.d. from three independent experiments. *P<0.0001.

• Supplemental Figure S2 -

  Fig. S2. (A) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, treated with 5 μM mitomycin C for 6 hours and incubated with 60 μg/ml cycloheximide for indicated times. (i) The amounts of p53 and GAPDH were determined by immunoblotting. (ii) The protein levels were quantified. (B) SH-SY5Y cells were pretreated with 10 μM roscovitine or Cdk2/5 inhibitor for 30 minutes, and then incubated with 2 mM SNP for the indicated times. 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. (C) SH-SY5Y cells were treated with 2 mM SNP for the indicated times, and then nuclear fractions were subjected to immunoblotting. Cdk5 kinase activity was determined by autoradiography using histone H1 as a substrate.

• Supplemental Figure S3 -

  Fig. S3. (A) H1299 cells were transfected with vectors encoding DsRed-p53, DsRed-p53 plus Hdm2, DsRed-p53 plus Hdm2 plus EGFP-Cdk5-p35, or DsRed-p53 plus Hdm2 plus EGFP-D144N-p35, as indicated. At 36 hours post-transfection the cells were analyzed by fluorescence microscopy. Hoechst staining was used to visualize cell nuclei. (B) HEK293T cells were transfected with vectors encoding Hdm2 or Cdk5-p25, as indicated. Nuclear and cytoplasmic fractions were prepared, and p53, Cdk5 and p25 were determined by western blotting using the indicated antibodies.

• Supplemental Figure S4 -

  Fig. S4. (A) SH-SY5Y cells were transiently transfected with Flag-tagged wild-type p53 (p53-WT) or Flag-tagged mutant p53 in which residues Ser15, Ser33 and Ser46 were changed to alanine (p53-SDM), treated with 5 μM mitomycin C for 6 hours and then incubated with 60 μg/ml cycloheximide for the indicated times. (i) The amounts of p53 and GAPDH were determined by immunoblotting. (ii) Protein levels were quantified. (B) SH-SY5Y cells were transfected with p53-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<0.001.