Kang et al. 10.1073/pnas.0506517102. |
Fig. 5. (A) Parental MDA-MB-231 cells were incubated with TGF-b for the indicated times. Total RNA was subjected to Northern blot analysis using IL-11 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes. (B) Several SCP cell lines derived from MDA-MB-231 were infected with the retroviruses expressing Smad4-targeting short-hairpin RNAs (shRNA) and shRNA-insensitive FLAG-tagged Smad4. Protein expression was assessed by direct immunoblotting of total lysates using the indicated antibodies. (C) Smad4-deficient MDA-MB-468 cells were transfected with 1 µg of pIL-11(–100)-Luc reporter plasmid (1), together with 0.5 µg of the indicated Smad expression plasmid (1), treated with or without TGF-b, and analyzed for luciferase activity. Data are the average of triplicate determinations ± SD. (D) MDA-MB-231 cells were incubated in the absence or presence of TGF-b for 2 h. Cell lysates were incubated with biotinylated oligonucleotides corresponding to the indicated IL-11 promoter probes. DNA-bound proteins were precipitated by streptavidin–agarose and detected by immunoblotting. A mutant c-myc TGF-b response element (mTIE) (2) was used as a negative control. (E) A549 cells were incubated with 100 nM TPA or 70 µM curcumim or with no additions for 30 min and then with 100 pM TGF-b for the indicated period. Total RNA was subjected to Northern blot analysis with the indicated probes.
1. Tang, W., Yang, L., Yang, Y. C., Leng, S. & Elias, J. A. (1998) J. Biol. Chem. 273, 5506–5513.
2. Chen, C. R., Kang, Y., Siegel, P. M. & Massague, J. (2002) Cell 110, 19–32.
Supporting Materials and Methods
Immunohistochemistry. Immunohistochemical analysis was performed with a Discovery XT System (Ventana Medical Systems, Tucson, AZ) using tissue sections blocked for 30 min in 10% normal goat serum (catalog no. S-1000, Vector Laboratories) and 2% BSA. Incubation with anti-phospho-Smad2 (Ser-465/467) primary antibody (catalog no. 3101, Cell Signaling Technology, Beverly, CA) at a 1:500 dilution was carried out for 3 h at room temperature followed by a 1-h incubation with biotinylated anti-rabbit secondary antibody at a 1:200 dilution (Vectastain ABC Kit Rabbit IgG, catalog no. PK-6101, Vector Laboratories) and DAB detection kit (Ventana Medical Systems) according to the manufacturer’s instructions.
Cell Culture and Retroviral Transduction.
Parental (American Type Culture Collection) MDA-MB-231 cell line and its various sublines, as well as the A549 cell line, were maintained in DMEM supplemented with 10% FBS, penicillin, streptomycin, and fungizone. Phoenix cells, a helper cell line for retrovirus production, were maintained in DMEM supplemented with 10% FBS, 1% glutamine, and antibiotics.Retroviruses expressing Smad4 short-hairpin RNA (shRNA), FLAG-Smad4M, or imaging proteins, were produced from amphotropic Phoenix packaging cell line. Phoenix cell transfections were performed using Lipofectamine (Invitrogen), according to the manufacturer’s instructions. Viruses were harvested 48 and 72 h after transfection, filtered, and used to infect MDA-MB-231 cell cultures in the presence of 5 mg/ml polybrene. Infected cells were selected by FACS for GFP-positive cells or by selection for puromycin or hygromycin resistance. To avoid clonal variations, we pooled at least 2,000 individual transfectants for each stable cell line produced by transduction.
Plasmids.
The minimal IL-11 promoter region containing the TATA box (–31 to +52) was cloned as a KpnI/BglI fragment into the corresponding sites in the pXP2-luc (American Type Culture Collection) to create pIL-11--TATA--Luc. Various IL-11 promoter regions immediately upstream of the TATA box were then inserted as BamHI/KpnI fragments upstream of the TATA box to generate a series of luciferase reporters controlled by different regions of the IL-11 promoter. Retroviral vectors that encode shRNAs against the hSmad4 transcript were generated by cloning suitable oligonucleotide sequences into the pSUPER-retro-puro vector (1). The coding strand of the Smad4 targeting shRNAs were GGATGAATATGTGCATGAC (Smad4-shRNA1) and GGTGTGCAGTTGGAATGTA (Smad4 shRNA2). A cDNA sequence encoding FLAG epitope-tagged hSmad4 was cloned to the BamHI/SalI sites of pBabe-hygro (2) to generate pBabe-hygro-FLAG-Smad4. Silent mutations were generated by site-directed mutagenesis in the coding sequence of Tyr-162 (from TAT to TAC) and Val-163 (from GTG to GTT) to create a shRNA-insensitive version of Smad4 expression plasmid pBabe-hygro-Flag-Smad4M.Luciferase Reporter Assays.
Luciferase reporter assays were performed as described in ref. 3. TGF-b1 (100 pM; R & D Systems), 10 mg/ml cycloheximide (Sigma), 100 nM TPA (Sigma), and 70 mM curcumin (Sigma) were used to treat cells in various assays. Northern blot analysis was carried out as described in ref. 3.ELISA Analysis.
The production and secretion of IL-11 in various sublines of MDA-MB-231 were determined in 24-h-conditioned media using commercially available IL-11 (R & D Systems) ELISA kits according to the manufacturer’s instructions.Transcriptomic Profiling and Clustering Analyses.
Tissue collection, RNA sample collection, and generation of biotinylated complementary RNA probe were carried out essentially as described in the standard Affymetrix (Santa Clara, CA) GeneChip protocol. Each sample was hybridized with an Affymetrix Human Genome U133A microarray for 16 h at 45°C. Absolute analysis of each chip and comparative analysis of TGF-b-treated samples with the untreated samples were carried out with <zcom>Affymetrix<zcomx> MICROARRAY SUITE 5.0 software. Genes whose expression level was changed by >2-fold with P < 0.001 were scored as TGF-b-regulated genes. Illustration of TGF-b gene responses in MDA-MB-231 and MCF-10A cell lines were produced with GENESPRING (Agilent Technologies, Palo Alto, CA) software.Electrophoretic Mobility Shift Assay.
Purified full-length Smad4 protein was used in this experiment. Complementary oligonucleotides corresponding to the wild-type IL-11 promoter and its mutants were annealed and end-labeled with g32P-ATP. The sequences for the probes are: 5'-GGGTGAGTCAGGATGTGTCAGGCCGGCCCTCCCCTGCCGCCTGCCCCCCGCCCGCCCGCCCCAGGCCCC-3' for wild type, 5'-GGGTTCTTCAGGATTGTTCAGGCCGGCCC TCCCCTGCCGCCTGCCCCCCGCCCGCCCGCCCCAGGCCCC-3' for mutant activator protein-1 (mAP1); 5'-GGC CGGCCCTCCCCTGCCGCCTGCCCCCCGCCCGCCCGCCCCAGGCCCC-3' for GC, 5'-GGGTGAGTCAGGATGTGTCA-3' for AP1, and 5'-GTAAGCCCGGCCAGCCGACC GGGGC3' for b-actin.The DNA–protein-binding reactions were performed and analyzed on a 5% nondenaturing gel. For supershift assessment, 1 ml of mouse monoclonal antibody (catalog no. 610843, BD transduction Laboratories) against Smad4 was preincubated with full-length recombinant His-Smad4 for 5 min on ice. DNA–protein complexes were visualized by autoradiography.
DNA Precipitation Assay.
DNA precipitation assays were carried out as described in ref. 4. The sequences of oligonucleotides used are as follows: 5'-GGGTGAGTCAGGATGTGTCAGGCCGGCCCTCCCCTGCCGCCTGCCCCCCGCCCGCCCGCCCCA-3' for wild type, 5'-GGGACAATCCGGACAATCCGGCCGGCCCTCCCCTGCCGCCTGCCCCCC GCCCGCCCGCCCCA-3' for mAP1, 5'-GGCCGGCCCTCCCCTGCCGCCTGCCCCCCGCCCGCCCGCCCCAGGCCCCC-3' for GC. 5'-GGGTGAGTCAGGATGTGTCAGGCCGGCCCTCCCCTGCCGCC-3' for AP1GC5', and 5'-GGGTGAGTCAGGATGTGTCATGCCCCCCGCCCGCCCGCCCCA-3' for AP1GC3'. Nucleotides that were mutated in the corresponding mutant probes were highlighted with italic letters. The sequence for TGF-b inhibitory element is reported in ref. 4.Intracardiac Injections.
Cells were harvested from subconfluent cell culture plates, washed with PBS, and resuspended at a concentration of 106 cells per ml of PBS. Of the suspended cells, 0.1 ml was injected into the left cardiac ventricle of 4-week-old, female BALB/c-nu/nu nude mice (National Cancer Institute, Bethesda) using 26-gauge needles as described in ref. 5. Mice were anesthetized with ketamine (100 mg per kg of body weight) and xylazine (10 mg per kg of body weight) before injection. A successful injection was characterized by the pumping of arterial blood into the syringe and by immediate bioluminescence imaging. All experiments using mice were performed under protocols approved by the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee.Radiographic Analysis of Bone Metastasis.
Development of bone metastases was monitored by x-ray radiography. Mice were anesthetized, arranged in prone position on single-wrapped films (X-Omat AR, Eastman Kodak), and exposed to an x-ray at 35 kV for 15 sec using a Faxitron instrument (Model MX-20, Faxitron, Buffalo, IL). Films were developed with a SRX-101A processor (Konica Minolta) and inspected for visible bone lesions.1. Brummelkamp, T. R., Bernards, R. & Agami, R. (2002) Cancer Cell 2, 243–247.
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3. Chen, C. R., Kang, Y. & Massagué, J. (2001) Proc. Natl. Acad. Sci. USA 98, 992–999.
4. Chen, C. R., Kang, Y., Siegel, P. M. & Massague, J. (2002) Cell 110, 19–32.
5. Yin, J. J., Selander, K., Chirgwin, J. M., Dallas, M., Grubbs, B. G., Wieser, R., Massague, J., Mundy, G. R. & Guise, T. A. (1999) J. Clin. Invest. 103, 197–206.