Expression analysis of murine genes using in situ hybridization with radioactive and nonradioactively labeled RNA probes.

The term in situ hybridization (ISH) refers to all methods allowing the detection of specific DNA (gene loci) or RNA (gene expression products) sequences, using molecular hybridization (base pairing) of labeled nucleic acid probes to target molecules within "intact" cell populations in tissue sections or whole organisms, cultured cells, or chromosomal spreads. For more than two decades, ISH has been one of the main approaches used to characterize gene expression patterns in all laboratory animal models, especially in the context of embryonic development, as well as in human tissue or cell samples for both research and diagnostic purposes. Here, we describe several ISH protocols applied to the analysis of mouse embryos and tissues; this organism has become a reference for mammalian experimental genetics. These protocols use in vitro transcribed RNAs as probes for detection. Radiolabeled probes (using 35S as a radioisotope) allow sensitive ISH on sections of paraffin-embedded material, whereas nonradioactively (digoxigenin) labeled probes can be used both for hybridization of whole embryos (whole-mount ISH) and frozen tissue sections

ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Abstract Background The ETS transcription factor ETV4 is involved in the main steps of organogenesis and is also a significant mediator of tumorigenesis and metastasis, such as in breast cancer. Indeed, ETV4 is overexpressed in breast tumors and is associated with distant metastasis and poor prognosis. However, the cellular and molecular events regulated by this factor are still misunderstood. In mammary epithelial cells, ETV4 controls the expression of many genes, MMP13 among them. The aim of this study was to understand the function of MMP13 during ETV4-driven tumorigenesis. Methods Different constructs of the MMP13 gene promoter were used to study the direct regulation of MMP13 by ETV4. Moreover, cell proliferation, migration, invasion, anchorage-independent growth, and in vivo tumorigenicity were assayed using models of mammary epithelial and cancer cells in which the expression of MMP13 and/or ETV4 is modulated. Importantly, the expression of MMP13 and ETV4 messenger RNA was characterized in 456 breast cancer samples. Results Our results revealed that ETV4 promotes proliferation, migration, invasion, and anchorage-independent growth of the MMT mouse mammary tumorigenic cell line. By investigating molecular events downstream of ETV4, we found that MMP13, an extracellular metalloprotease, was an ETV4 target gene. By overexpressing or repressing MMP13, we showed that this metalloprotease contributes to proliferation, migration, and anchorage-independent clonogenicity. Furthermore, we demonstrated that MMP13 inhibition disturbs proliferation, migration, and invasion induced by ETV4 and participates to ETV4-induced tumor formation in immunodeficient mice. Finally, ETV4 and MMP13 co-overexpression is associated with poor prognosis in breast cancer. Conclusion MMP13 potentiates the effects of the ETV4 oncogene during breast cancer genesis and progression

Additional file 7: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S5. Expression of ETV4 in MMT-shMMP13-repressing cells. a Relative ETV4 mRNA expression in the MMT-ETV4 + shCtrl and MMT-ETV4 + shMMP13 cells determined by real-time PCR and normalized to cyclophilin A levels. mRNA expression in MMT-Ctrl + shCtrl cells was arbitrarily = 1. Error bars indicate SD. The results were not statistically significant. b Western blot analysis of ETV4 protein expression (61 kDa) in the MMT-ETV4 + shCtrl and MMT-ETV4 + shMMP13 cells. GAPDH expression served as the loading control. (PDF 71 kb

Additional file 8: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S6. The repression of MMP13 reduces the anchorage-independent growth capacity of MMT-ETV4-overexpressing cells. a Relative MMP13 mRNA expression in the transiently transfected MMT-siCtrl and MMT-siMMP13 cells determined by real-time PCR and normalized to cyclophilin A levels. mRNA expression in MMT-siCtrl cells was arbitrarily = 1. Error bars indicate SD. ****P ≤ 0.0001. b Anchorage-independent growth. MMT-ETV4-siCtrl and MMT-ETV4-siMMP13 cells were cultured for 10 days in soft agar. This histogram represents the number of clones counted for experimental time points. Soft agar assays were conducted three times in triplicate. Magnification × 5. Error bars indicate SD. ****P ≤ 0.0001. (PDF 45 kb

Additional file 10: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S8. Metastasis-free survival analysis from the publicly available NKI datasets of breast tumors. a Metastasis-free survival (MFS) curves for patients with breast tumors according to Low-ETV4 (n = 12), High-ETV4 and Low-MMP13 (n = 243), or High-ETV4 and High-MMP13 (n = 9) mRNA levels. ****P ≤ 0.0001. b Metastasis-free survival (MFS) curves for breast tumor patients according to Low-MMP13 (n = 255) or High-MMP13 (n = 9) mRNA levels. ****P ≤ 0.0001. c Metastasis-free survival (MFS) curves for patients with breast tumors according to Low-ETV4 (n = 13) and High-ETV4 (n = 251) mRNA levels. ****P ≤ 0.0001. (PDF 19 kb