HEK293 cells and human breast cancer MCF-7 cells, ZR-75-1 cells, were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The HCT116 p53−/− and HCT116 p53+/+ cell lines were gifts from Dr. Vogelstein (Johns Hopkins University, USA)  and Dr. Zhihua Liu (Chinese Academy of Medical Sciences and Peking Union Medical College, China) . MCF-7 cells were grown in MEM medium that was supplemented with 10% foetal bovine serum, 1% non-essential amino acids and 10 μg/ml insulin. ZR-75-1 cells were grown in RPMI-1640 medium with 10% foetal bovine serum. HEK293 cells, HCT116 p53−/− and p53+/+ cells were maintained in DMEM that was supplemented with 10% foetal bovine serum. All of the cells were maintained in a humidified atmosphere that contained 5% CO2 at 37°C.
Plasmid construction and mutagenesis
The −1807/+60, −979/+60, −640/+60, −294/+60 and −116/+60 fragments of the human IBP gene (relative to the transcriptional start site) were amplified from the genomic DNA of MCF-7 cells by PCR using KOD polymerase (Toyobo). These amplified fragments were inserted into the Kpn I and Hind III restriction sites of the pGL3-basic vector (Promega). The wild-type p53 expression plasmid, pCMV-p53, and the p53 mutant plasmid, pCMV-p53R175H, were kindly provided by Dr. Vogelstein (Johns Hopkins University, USA). TaKaRa MutanBEST kit (TaKaRa) was used to introduce the p53 binding site into the IBP promoter deletion mutant. The following mutagenic primers were used: forward 5′-CGGGAGCCACGTGGATACAG-3′, reverse 5′-TTTTAGAAGCCTCCTCAGACCC-3′. The pEGFP-C1-IBP expression plasmid was a gift from Dr. Alessandra B. Pernis (Columbia University, USA). All of the constructs were confirmed by DNA sequencing.
Adenovirus infection and cell treatment
Adenovirus(Ad)-p53 was purchased from Shenzhen SiBiono GeneTech Co. . Ad-GFP was purchased from Shanghai Sunbio Medical Biotechnology Co. The cells were treated with different concentrations of doxorubicin (Sigma-Aldrich) for 8 h, Nutlin-3 (Beyotime) for 24 h and pifithrin-α (Beyotime) for 24 h. The cisplatin (Sigma-Aldrich) concentrations and experimental details are described in the text and figure legends. The cells were treated with Ly294002 (Beyotime) or wortmannin (Beyotime) for 24 h.
To knockdown IBP expression, double-stranded DNA oligonucleotides (forward, 5′-TGCTGTTCATCTGGACATTCCAGTGTGTTTTGGCCACTGACTGACACACTGGAGTCCAGATGAA-3′ and reverse, 5′-CCTGTTCATCTGGACTCCAGTGTGTCAGTCAGTGGCCAAAACACACTGGAATGTCCAGATGAAC-3′) were subcloned into pcDNA™6.2-GW/EmGFPmiR (Invitrogen) using the BLOCK-iT Pol II miR RNAi Expression Vector Kit (Invitrogen). The RNAi plasmid or control plasmid, which contained a non-specific sequence, was transfected into MCF-7 cells. Lipofectamine 2000 (Invitrogen) was used as the transfection reagent. The growth medium was supplemented with blasticidin (10 μg/ml, Invitrogen), which was used to select for blasticidin-resistant transfectants. For the p53 knockdown, double-stranded DNA oligonucleotides (forward, 5′-CCGGGACTCCAGTGGTAATCTACTTCAAGAGAGTAGATTACCACTGGAGTCTTTTTG-3′ and reverse, 5′-AATTCAAAAAGACTCCAGTGGTAATCTACTCTCTTGAAGTAGATTACCACTGGAGTC-3′) were subcloned into pMagic 1.1 and packaged into lentivirus particles (Shanghai Sunbio Medical Biotechnology Co.). One day after infection, the cell-growth medium was supplemented with puromycin (2 μg/ml, Invitrogen) to select stable transfectants.
Luciferase reporter assays
Luciferase reporter assays were performed using the Dual-Luciferase® Reporter Assay System (Promega). Cells were seeded in 24-well plates (1.0 × 105 cells/well) and transfected together with a promoter-reporter gene vector and the pRL-TK Renilla luciferase vector. After 48 h of transfection, the cells were harvested and analysed according to the manufacturer’s instructions. The luciferase activities were normalised to the Renilla luciferase activity of the internal control.
Cell lysates were prepared in RIPA buffer (Beyotime). Whole-cell lysates were separated on a 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked for 1 h at 37°C in 5% non-fat milk/TBST and were then incubated with primary antibodies overnight at 4°C. Antibodies against IBP (produced as described previously) , p53 (sc-126, Santa Cruz), p21 (3733–1, Epitomics), PARP (9532, Cell Signaling), phospho-p53(Ser15) (9284, Cell Signaling), Bcl-2 (1017–1, Epitomics), Bax (AB026, Beyotime), phospho-AKT(Ser473) (4060, Cell Signaling), AKT(4691, Cell Signaling),phospho-MDM2(Ser166) (3521, Cell Signaling), MDM2(sc-965, Santa Cruz) and GAPDH (AG019, Beyotime) were used. The membrane was then rinsed in TBST and incubated with various secondary antibodies for 2 h at 25°C. Immunoreactive bands were visualised with a chemiluminescent HRP substrate (Millipore).
Total RNA was isolated using TRIzol (Invitrogen), and 1 μg of isolated RNA was reverse transcribed to generate cDNAs (TaKaRa). Amplification was performed by using SYBR Premix Ex Taq II (TaKaRa). The primers used for amplification included the following: IBP forward, 5′-GAGGGCTGACGAGGATGTGG-3′ and reverse, 5′-GCTGGTGACCGGACGCTTAT-3′; and GAPDH forward, 5′-AATCCATCACCATCTTCCA-3′ and reverse, 5′-TGGACTCCACGACGTACTCA-3′. GAPDH mRNA levels were determined as an internal control.
Electrophoretic mobility shift assays (EMSA)
Nuclear extracts were prepared in hypertonic buffer (420 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, 25% glycerol, 5 μg/ml aprotinin, 5 μg/ml phenanthroline, 3 μg/ml pepstatin A and 20 mM HEPES). Double-stranded oligonucleotide probes that were derived from the IBP gene promoter (sense strand, 5′-TAAAAGGGCCTCACATGCCCCGGGAGCCAC-3′) and p21 gene promoter (sense strand, 5′-GGAAGAAGACTGGGCATGTCTGGGCAGAGA-3′)  were labelled with γ-32P-ATP using T4 polynucleotide kinase. The nuclear extracts (8 μg) were incubated with the probe for 30 min at 30°C. The protein-DNA complexes were resolved using non-denaturing PAGE and were detected by autoradiography. For the cold probe competition assay, unlabelled probe was added to the nuclear protein extracts one hour before the detection was performed. In the supershift assay, 1 μl of an anti-p53 antibody (sc-126x, Santa Cruz) was incubated with the nuclear extracts for 1 h at room temperature prior to the addition of the radiolabeled probe and the implementation of PAGE.
Chromatin immunoprecipitation assay (ChIP)
The ChIP assays were performed using an EZ-ChIPTM Chromatin Immunoprecipitation Kit (Upstate) following the manufacturer’s instructions. Briefly, cells were crosslinked with 1% formaldehyde and a p53 antibody (sc-126, Santa Cruz) or control IgG, which was used to precipitate the crosslinked protein/chromatin. The DNA fragments were analysed using PCR with a primer set (forward, 5′-TTTTCCCTCAGCAAGCTGCGTCTGG-3′ and reverse, 5′-CTGCATGGGAACTGGGGACCAACTCT-3′) that was designed to amplify the −305 to −150 region of the IBP gene that harbours p53-binding site.
Cell survival assays
A cell survival assay was performed in triplicate with a Cell Counting Kit-8 (CCK-8, Beyotime). The cells were seeded in 96-well plates at 5 × 103 cells/well (100 μl/well) 24 h before the cisplatin treatment. The culture medium was then replaced with fresh medium that contained different concentrations of cisplatin, which ranged from 0 to 32 μg/ml, and the cells were cultured in this medium for 24 h. Following the incubation, 10 μl of CCK-8 solution was added to each well, and after 1 h, the absorbance value of each well was read at 450 nm. The cell growth rate was calculated as the ratio of the absorbance of the experimental well to that of the blank well. The IC50 values (the drug concentration that results in a 50% absorbance reduction compared to the control) were calculated.
Annexin V-PI flow cytometry assay
Flow cytometry assay was performed by using Caliber II sorter and Cell Quest FACS system (BD Biosciences). Alexa fluor 647 conjugated Annexin V (invitrogen) and PI (Invitrogen) was incubated for 15 min according to the manufacturer’s protocol. About 104 cells were measured per sample.