SEARCH

SEARCH BY CITATION

Fig. S1. PCR primers and oligomers used in this study.

Fig. S2. (a) Quantitative RT-PCR of crystallin alpha B (CRYAB) using transiently knocked down p53 MCF-7 breast cancer cells. IB, immunoblotting. (b) MCF-7 cells were grown to 60% confluence on a 6-well plate and 0.5 μg p53 siRNA or control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were transfected according to the manufacturer’s protocol. After 24 h, these cells were treated or not treated with 60 J/m2 of UV. A further 24 h after incubation, cells were harvested and mRNA extracted.

Fig. S3. Gel shift assay for p53RE1 in crystallin alpha B (CRYAB). Nuclear extracts were prepared from SF126-tet-p53 cells with or without 5 ng/mL doxycycline using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL, USA) according to the manufacturer’s protocol. The extracts were incubated at room temperature for 30 min in DNA binding buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM DTT, 0.5% Tween-20] containing IRDye 700 (LI-COR Bioscience, Lincoln, NE, USA) labeled p53RE1 double-stranded oligomers annealed by p53RE1s and p53RE1as. In some experimental condition, non-labeled p53RE1 double stranded DNA oligomer or non-labeled p53RE1mt double stranded DNA oligomer (a product of annealing of p53RE1 mutant-s with p53RE1 mutant-as) were added to the extract with monoclonal anti-p53 antibodies, Pab421 and/or Pab1801. These oligomers are listed in Supporting information Fig.  S1. The reactions were loaded on a 4% PAGE using 0.5 × TBE buffer and electrophoresed for 2 h at 120 V. The gels were scanned by the Odyssey infrared imaging system (LI-COR Bioscience, Lincoln, NE, USA). Using SF126-tet-p53 cells, nuclear extracts were prepared under these conditions with or without p53 expression. The lower triangle indicates p53-p53RE1-Pab421 complex (sifted band). The upper triangle indicates p53-p53RE1-Pab421-Pab1801 complex (super-sifted band).

Fig. S4. Immunostaining for p53 and αB-crystallin. SF126 glioblastoma cells 1 × 104 were cultured directly on poly-D lysine coated Lab-Tek Chamber Slide (Nalge Nunc, Rochester, NY, USA) and transfected with pCR259-p53 (50 ng) or pCR259-cryab (150 ng). After 24 h, the cells were fixed with methanol-acetone (1:1) for 10 min at –20°C. Fixed cells were washed three times with PBS containing 0.05% Tween-20 (PBS-T). After blocking with PBS containing 5% skim milk at room temperature for 30 min, cells were incubated with anti-p53 (1:100 dilution) (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-αB-crystallin (1:100 dilution) (SPA-223; Stressgen Biotechnology, Ann Arbor, MI, USA) antibodies. After washing with PBS-T, cells were incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:400 dilution; Molecular Probes, Invitrogen, Carlsbad, CA, USA) and FITC-conjugated goat anti-mouse IgG (1:400 dilution; Molecular Probes). The stating cell was visualized on an LSM5 Pascal (Carl Zeiss, G&oumlet;ottingen, Germany). Green, p53; red, αB-crystallin.

Fig. S5. Overexpressed αB-crystallin stabilized p53. pCR259-p53 or pCR259-cryab was transfected in SF126 glioblastoma cells according to the indicated amount of plasmid with 100 ng of GFP expression plasmid. After 24 h transfection, cell lysates were immunoblotted using p53 and αB-crystallin or GFP antibodies. The relative expression level of p53 or αB-crystallin compared with GFP was analyzed using Kodak 1D Image Analysis Software (lower panel).

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

FilenameFormatSizeDescription
CAS_1316_sm_f1.xls31KSupporting info item
CAS_1316_sm_f2.tif453KSupporting info item
CAS_1316_sm_f3.tif3814KSupporting info item
CAS_1316_sm_f4.tif2641KSupporting info item
CAS_1316_sm_f5.tif27668KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.