6. End of the experiment: in this chapter, we describe the realization of four successive cycles of EMT/MET. It is up to the user
to define the number of cycles that he/she wishes to achieve
according to his hypotheses to be tested. It must be kept in
mind that the duration of the experiment is proportional to the
number of cycles (4 cycles ¼ 1 month, 8 cycles ¼ 2 months,
etc.).
7. Choice of EMT and MET markers: we selected well-known
EMT and MET markers (EMT-TFs like SNAIL and ZEB,
E-cadherin, β-Catenin, Fibronectin and Vimentin) combined
with different methods to evaluate their expression level fluctuation. This gave us a fairly robust validation of the effectiveness of the EMT/MET cycles. Interestingly, the oscillations are
clearly visible for both proteins and transcripts.
Acknowledgements
We acknowledge all the members of UMRS935 for technical assistance and scientific daily exchanges.
This work was funded by Inserm, University Paris Sud, Association
Institut de Cance ´rologie et d’Immunoge ´ne ´tique (ICIG) and
Vaincre le Cancer-NRB. E.H. post-doctoral fellowship was granted
by Vaincre le Cancer-NRB and the University Paris-Saclay (Project
BioTherAlliance). C.D. Ph.D. fellowship was granted by University
Paris-Saclay.
Author contribution: C.D., W.T., E.H. and H.A. performed the
experiments.
C.D., E.H., W.T., A.B.G. and H.A. designed the experimental
strategy and analysed the results.
C.D., E.H., W.T., A.B.G. and H.A. wrote the manuscript.
References
1. Nieto MA, Huang RY, Jackson RA, Thiery JP
(2016) EMT: 2016. Cell 166:21–45
2. Hay ED (1968) Organization and fine structure of epithelium and mesenchyme in the
developing chick embryo. In: Fleischmajer R,
Billingham RE (eds) Epithelial-mesenchymal
interactions. Williams & Wilkins, Baltimore,
MD, pp 31–55
3. Acloque H, Adams MS, Fishwick K, BronnerFraser M, Nieto MA (2009) Epithelialmesenchymal transitions: the importance of
changing cell state in development and disease.
J Clin Invest 119:1438–1449
4. Acloque H, Ocan ˜a OH, Matheu A, Rizzoti K,
Wise C, Lovell-Badge R, Nieto MA (2011)
Reciprocal repression between Sox3 and snail
transcription factors defines embryonic territories at gastrulation. Dev Cell 21:546–558
5. del Barrio MG, Nieto MA (2002) Overexpression of snail family members highlights their
ability to promote chick neural crest formation.
Development 129:1583–1593
6. Gros J, Scaal M, Marcelle C (2004) A two-step
mechanism for myotome formation in chick.
Dev Cell 6:875–882
7. Thiery JP, Acloque H, Huang RY, Nieto MA
(2009) Epithelial-mesenchymal transitions in
development and disease. Cell 139:871–890
8. Ocan ˜a OH, Co ´ rcoles R, Fabra A, MorenoBueno G, Acloque H, Vega S, Barrallo350
Cecile Davaine et al.
to define the number of cycles that he/she wishes to achieve
according to his hypotheses to be tested. It must be kept in
mind that the duration of the experiment is proportional to the
number of cycles (4 cycles ¼ 1 month, 8 cycles ¼ 2 months,
etc.).
7. Choice of EMT and MET markers: we selected well-known
EMT and MET markers (EMT-TFs like SNAIL and ZEB,
E-cadherin, β-Catenin, Fibronectin and Vimentin) combined
with different methods to evaluate their expression level fluctuation. This gave us a fairly robust validation of the effectiveness of the EMT/MET cycles. Interestingly, the oscillations are
clearly visible for both proteins and transcripts.
Acknowledgements
We acknowledge all the members of UMRS935 for technical assistance and scientific daily exchanges.
This work was funded by Inserm, University Paris Sud, Association
Institut de Cance ´rologie et d’Immunoge ´ne ´tique (ICIG) and
Vaincre le Cancer-NRB. E.H. post-doctoral fellowship was granted
by Vaincre le Cancer-NRB and the University Paris-Saclay (Project
BioTherAlliance). C.D. Ph.D. fellowship was granted by University
Paris-Saclay.
Author contribution: C.D., W.T., E.H. and H.A. performed the
experiments.
C.D., E.H., W.T., A.B.G. and H.A. designed the experimental
strategy and analysed the results.
C.D., E.H., W.T., A.B.G. and H.A. wrote the manuscript.
References
1. Nieto MA, Huang RY, Jackson RA, Thiery JP
(2016) EMT: 2016. Cell 166:21–45
2. Hay ED (1968) Organization and fine structure of epithelium and mesenchyme in the
developing chick embryo. In: Fleischmajer R,
Billingham RE (eds) Epithelial-mesenchymal
interactions. Williams & Wilkins, Baltimore,
MD, pp 31–55
3. Acloque H, Adams MS, Fishwick K, BronnerFraser M, Nieto MA (2009) Epithelialmesenchymal transitions: the importance of
changing cell state in development and disease.
J Clin Invest 119:1438–1449
4. Acloque H, Ocan ˜a OH, Matheu A, Rizzoti K,
Wise C, Lovell-Badge R, Nieto MA (2011)
Reciprocal repression between Sox3 and snail
transcription factors defines embryonic territories at gastrulation. Dev Cell 21:546–558
5. del Barrio MG, Nieto MA (2002) Overexpression of snail family members highlights their
ability to promote chick neural crest formation.
Development 129:1583–1593
6. Gros J, Scaal M, Marcelle C (2004) A two-step
mechanism for myotome formation in chick.
Dev Cell 6:875–882
7. Thiery JP, Acloque H, Huang RY, Nieto MA
(2009) Epithelial-mesenchymal transitions in
development and disease. Cell 139:871–890
8. Ocan ˜a OH, Co ´ rcoles R, Fabra A, MorenoBueno G, Acloque H, Vega S, Barrallo350
Cecile Davaine et al.