Abe et al. 10.1073/pnas.0712216105. |
SI Materials and Methods
Cells, Plasmids and Antibodies.
C3H10T1/2 cells and human cancer cell lines were purchased from the Health Science Research Resources Bank. C3H10T1/2 cells were maintained in Basal Medium Eagle (BME) (Sigma) supplemented with 10% FBS. YMB1E cells were maintained in RPMI1640 medium supplemented with 10% FBS. Saos-2 cells and MEFs were maintained in DMEM supplemented with 10% FBS. MRK-nu-1 cells were maintained in DMEM/F-12 medium (GIBCO) supplemented with 10% FBS. The expression vectors used for epitope-tagged (with the gD epitope from HSV) human SMO-WT, -M1 and -M2 have been described elsewhere (1). Dominant-negative Akt has been described (2). All cDNAs were inserted into the retroviral vector pBabe puro (3). Gli1 and Gli3 cDNAs containing HA-tagged sequences were inserted into the pEF vector (4). Adenovirus E1A was inserted into the pBabe hygro vector (3). Anti-p53 (FL-393 and Pab246; Santa Cruz Biotechnology), anti-Mdm2 (2A10; Calbiochem), anti-p21 (Santa Cruz Biotechnology), anti-cyclin D1 (72G13; Santa Cruz Biotechnology), anti-cyclin E (E-4; Santa Cruz Biotechnology), anti-ATM (Santa Cruz Biotechnology), anti-phospho-ATM Ser-1981 (Cell Signaling Tecnology), anti-phospho-Histone H2AX Ser-139 (Upstate Biotechnology), anti-Flag (M2; Sigma), anti-Myc (9E10; Santa Cruz Biotechnology), anti-HA (16B12; Covance), anti-Akt (Santa Cruz Biotechnology), and anti-phospho-Akt Ser-473 (Cell Signaling Technology) antibodies were used according to the manufacturers' recommendations. The anti-phosoho-Hdm2 (Ser-166) antibody has been described in ref. 5, and the anti-phosoho-Hdm2 (Ser-186) antibody was prepared by using the same method.Immunoprecipitation and Immunoblotting.
To detect the amount of endogenous or transiently produced p53 protein, cells were lysed in RIPA buffer (1% Nonidet P-40, 0.5% sodium-deoxycholate, 0.1% SDS, 10 mM Tris×HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, and a protease inhibitor mixture [Nacalai]). To detect binding between endogenous p53 and Mdm2, or phosphorylation of Mdm2 and Akt, cells were lysed in lysis buffer [0.5% Nonidet P-40, 150 mM NaCl, 10 mM Tris×HCl (pH 8.0), 1 mM EDTA, 1 mM NaF, 1 mM orthovanadate 0.1 mM DTT, and a protease inhibitor mixture]. Immunoprecipitation was carried out as described in ref. 6. Total cell lysate protein was quantified by using a Protein Assay kit (Bio-Rad), and an equal amount of protein was used for immunoprecipitation and immunoblotting.Detection of p53 Ubiquitination.
To detect the ubiquitinated form of p53 in cells, 1 ´ 106 C3H10T1/2 cells were transfected with 1.8 mg of HA-tagged p53, 2 mg of Flag-tagged ubiquitin and 0.2 mg of SMO-M1 or -M2 expression plasmid. Twenty four hours after transfection, the cells were treated with 50 mM MG132 (Calbiochem) for 4 h, and then lysed in 25 mM MG132-containing RIPA buffer. Cell extracts were immunoprecipitated with anti-HA antibody and analyzed by immunoblotting. As shown in Fig. 2e, cells were treated with sonic hedgehog N-terminal peptide (Shh-N) for 48 h, and then lysed in RIPA buffer. The cells were treated with 50 mM MG132 for 4 h before being harvested. Cell extracts were immunoprecipitated with anti-p53 (FL-393) antibody and analyzed by immunoblotting with anti-p53 (Pab246) antibody.Subcellular localization of p53.
For immunofluorescence staining, C3H10T1/2 cells were grown on 96-well glass-bottom plates (IWAKI). To assess the subcellular localization of p53 in Smo mutant-expressing cells, 1 ´ 103 C3H10T1/2 cells were transfected with 5 ng of pEF-p53 and 45 ng of pRK7-gD-SMO M1 or M2. Twenty four hours after transfection, the cells were fixed with 4% paraformaldehyde for 15 min at 37°C, and permeabilized with 0.2% Triton X-100 for 7 min at room temperature. Fixed cells were then incubated in 1% BSA-PBS for 30 min followed by anti-p53 (FL-393) or monoclonal anti-gD antibody (7) for 30 min at room temperature. After washing with PBS, fixed cells were incubated for 30 min at room temperature with Alexa Fluor 488- or Alexa Fluor 568-conjugated anti-mouse IgG or anti-rabbit IgG (Molecular Probes) antibody. Finally, the cells were counterstained with DAPI to visualize their nuclei. Immunofluorescence was recorded by using an immunofluorescence microscope (OLYMPUS). Images were captured by using a Penguin 150CL camera (Pixera) and imaging software (Pixera).Apoptosis Detection.
E1A- and Smo-expressing MEFs (2 ´ 105) were treated with 0.6 mg/ml adriamycin for 24 h. Trypan blue dye exclusion analysis was performed to assess apoptosis. Cell viability was measured by using a Vi-Cell image analyzer (Beckman).RNAi.
A specific shRNA against Mdm2, a 64-base pair oligonucleotide containing a target sequence for Mdm2, and a control shRNA, were synthesized and cloned into pSUPER retro (Oligoengine). The oligonucleotides used were: sense-GATCCCCGGAACAAGAGACTCTGGTTTTCAAGAGAAACCAGAGTCTCTTGTTCCTTTTTGGAAA; antisense-AGCTTTTCCAAAAAGGAACAAGAGACTCTGGTTTCTCTTGAAAACCAGAGTCTCTTGTTCCGGG. The underlined sequences represent the linker regions and the italic sequences represent hairpin loop regions. p53 siRNA was expressed by using pSUPER retro as described in ref. 8.Colony Formation Assay.
p53-/- mice were purchased from Taconic. Animal experiments were reviewed by the Ethics Committee on Animal Experiments of Nippon Medical School and were carried out in accordance with the Guidelines for Animal Experiments of Nippon Medical School and the guidelines of The Law and Notification of the Japanese government. MEFs were prepared as described in ref. 4, and used in a colony formation assay. A bottom layer of 0.5% (wt/vol) agarose in normal medium was prepared in 35-mm culture plates. A top layer of 0.6% agarose containing 1 ´ 104 infected cells was also plated.1. Xie J, et al. (1998) Activating Smoothened mutations in sporadic basal-cell carcinoma. Nature 391:90-92.
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