Article Figures & Data
Figures
- Figure 1.
TGF-β1 induces Brachyury and EMT in human carcinoma cells. A, bright-field images of DU145 cells left untreated or treated for 72 hours with increasing concentrations of TGF-β1. Expression of E-cadherin and fibronectin in DU145 cells treated with TGF-β1 for 72 hours by (B) real-time PCR or (C) Western blot analysis. D, Brachyury expression in DU145 cells treated with TGF-β1 by real-time PCR or (E) Western blot analysis. Real-time PCR analysis of Brachyury expression in human prostate (F) and lung cancer (G) cell lines treated with indicated concentrations of TGF-β1 for 72 hours. Error bars indicate SEM for triplicate measurements; *, P < 0.05; **, P < 0.005; ***, P < 0.001.
- Figure 2.
TGF-β1–mediated Brachyury upregulation is relevant to tumor invasiveness. A, immunofluorescent analysis of Brachyury expression in DU145 cells treated as indicated for 72 hours. Green corresponds to Brachyury expression; blue indicates 4′, 6-diamidino-2-phenylindole (DAPI)-stained nuclei (magnification, ×40). B, DU145 cells treated as indicated for 48 hours were evaluated for cell migration and invasion. C, DU145 cells were left untransfected or transfected with control- versus Brachyury-specific siRNA for 24 hours and subsequently left untreated or treated for 48 hours with TGF-β1. At the end of the cytokine treatment, cells were harvested for Brachyury mRNA analysis or (D) invasion assays. E, real-time PCR analysis of Brachyury, Snail, and Slug in DU145 cells treated for 72 hours as indicated. Expression of Snail and Slug by real-time PCR (F) or Western blot analysis (G) in DU145 cells untransfected versus cells transfected with control- versus Brachyury-specific siRNA and subsequently incubated with TGF-β1 for 48 hours. Error bars indicate SEM for triplicate measurements. *, P < 0.05; **, P < 0.005; ***, P < 0.001, ****; P < 0.0001.
- Figure 3.
Brachyury induces TGF-β secretion. ELISA for TGF-β1 in culture supernatants of human cancer cell pairs obtained by (A) Brachyury overexpression in DU145 cells (pBrachyury) or (B) via stable silencing with a Brachyury-specific shRNA (Brac.shRNA) in SW620 and H460 cells. C, levels of TGF-β1 secreted by H460 cells silenced for Brachyury expression and subsequently transfected with an empty vector (pcDNA) or a pBrachyury vector. D, immunohistochemical analysis of TGF-β1 in xenografts of H460 control.shRNA versus H460 Brachyury.shRNA cells. Brown corresponds to TGF-β1 expression; slides were counterstained with hematoxylin. (magnification: left, ×10; right, ×20). E, secretion of TGF-β2 in culture supernatants of indicated tumor cells. Error bars indicate SEM for triplicate measurements. *, P < 0.05; **, P < 0.005; ***, P < 0.001.
- Figure 4.
Brachyury activates TGF-β1 promoter transcription. A and B, real-time PCR analysis of TGF-β1 in tumor cells with various levels of Brachyury. C, TGF-β1 promoter reporter activity normalized to GAPDH promoter in H460 control.shRNA versus Brachyury.shRNA. Error bars indicate SEM for triplicate measurements. *, P < 0.05; **, P < 0.005.
- Figure 5.
SD-208 reverses TGF-β1–induced EMT. A, chemical structure of SD-208. B, expression of E-cadherin, fibronectin, and Brachyury mRNA in DU145 cells untreated (−) or pretreated with SD-208 for 30 minutes followed by treatment with TGF-β1 for 72 hours. C, Western blot analysis of total Smad-2, phospho-Smad-2, and β-actin in protein lysates from DU145 cells pretreated with or without SD-208 for 30 minutes in the presence or absence of TGF-β1 for an additional 30 minutes. D, cell migration and ECM invasion assay with DU145 cells treated with TGF-β1 for 48 hours in the absence or presence of indicated doses of SD-208. Results from a representative experiment are shown. E, Western blot analysis for ZO-1, fibronectin, and β-actin in H460 cells treated for 72 hours as indicated. F, immunofluorescent analysis of Plakoglobin and fibronectin (green) in H460 cells; blue corresponds to nuclei stained with DAPI (original magnification, ×40). G, cell migration and ECM invasion assay with H460 cells treated as indicated for 48 hours. Error bars indicate SEM for triplicate measurements; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. phospho-Smad-2, P-Smad-2.
- Figure 6.
SD-208 decreases Brachyury expression and enhances chemotherapy sensitivity of human carcinoma cells. A, H460 cells were treated with increasing concentrations of SD-208 for 72 hours and evaluated for Brachyury and β-actin expression by Western blot analysis. Bottom graph shows Brachyury levels normalized to β-actin. B, chemical structure of chemotherapeutics used. C, H460 cells were left untreated (0) or pretreated for 48 hours with indicated concentrations of SD-208 and subsequently exposed to chemotherapeutics (165/0.12 ng/mL cisplatin/vinorelbine, 165 ng/mL cisplatin, 0.12 ng/mL vinorelbine, and 0.07 ng/mL docetaxel). Survival was evaluated after 4 days by the MTT assay. D, expression of Brachyury and GAPDH in indicated tumor cells incubated for 48 hours with increasing doses of SD-208. E, survival of H460 control.shRNA and Brachyury.shRNA cells pretreated with indicated doses of SD-208 and subsequently treated as above with cisplatin/vinorelbine. Cis, cisplatin; vin, vinorelbine (ng/mL).
Additional Files
Files in this Data Supplement:
- Supplementary Figure 1 - PDF file - 89K, TGF-β1 secretion in LNCap and H520 cells.
- Supplementary Figure 2 - PDF file - 326K, Treatment with SD-208 reverts the mesenchymal phenotype of colon carcinoma cell lines.
Volume 12, Issue 9
