Immunofluorescence Analysis by Confocal Microscopy for Detecting Endogenous FOXO

Part of the Methods in Molecular Biology book series (MIMB, volume 1890)


Cancer cells are known to inactivate tumor suppressor proteins by triggering their anomalous subcellular location. It has been well established that the aberrant location of FOXO proteins is linked to tumor formation, progression of the same, or resistance to anti-neoplastic treatment. Furthermore, the abnormal location of FOXO has also been considered a potential biomarker for diabetic complications or longevity in different organisms. Here, we describe the immunodetection of endogenous FOXO by confocal microscopy, which can be used as a chemical tool to quantify FOXO expression levels, its cellular location, and even its active/inactive forms with relevant antibodies.

Key words

Biomarker FOXO proteins Nuclear translocation Immunodetection Confocal microscopy 


  1. 1.
    Elias JM (1999) Immunohistochemistry: a brief historical perspective. J Histotechnol 22:163–167. CrossRefGoogle Scholar
  2. 2.
    Lehtinen MK, Yuan Z, Boag PR et al (2006) A conserved MST-FOXO signaling pathway mediates oxidative-stress responses and extends life span. Cell 125:987–1001. CrossRefPubMedGoogle Scholar
  3. 3.
    Liu P, Begley M, Michowski W et al (2014) Cell-cycle-regulated activation of Akt kinase by phosphorylation at its carboxyl terminus. Nature 508:541–545. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Hofman FM, Taylor CR (2001) Immunohistochemistry. Curr Protoc Immunol.
  5. 5.
    Yang J-Y, Zong CS, Xia W et al (2008) ERK promotes tumorigenesis by inhibiting FOXO3a via MDM2-mediated degradation. Nat Cell Biol 10:138–148CrossRefGoogle Scholar
  6. 6.
    Duraiyan J, Govindarajan R, Kaliyappan K, Palanisamy M (2012) Applications of immunohistochemistry. J Pharm Bioallied Sci 4:S307–S309. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Blagosklonny MV (2012) Common drugs and treatments for cancer and age-related diseases: revitalizing answers to NCI’s provocative questions. Oncotarget 3:1711–1724. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Kau TR, Way JC, Silver PA (2004) Nuclear transport and cancer: from mechanism to intervention. Nat Rev Cancer 4:106–117CrossRefGoogle Scholar
  9. 9.
    Seoane J, Le H-V, Shen L, Anderson SA, Massagué J (2004) Integration of smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 117:211–223. CrossRefPubMedGoogle Scholar
  10. 10.
    Garrett JT, Olivares MG, Rinehart C et al (2011) Transcriptional and posttranslational up-regulation of HER3 (ErbB3) compensates for inhibition of the HER2 tyrosine kinase. Proc Natl Acad Sci 108:5021–5026. CrossRefPubMedGoogle Scholar
  11. 11.
    Kau TR, Schroeder F, Ramaswamy S et al (2003) A chemical genetic screen identifies inhibitors of regulated nuclear export of a Forkhead transcription factor in PTEN-deficient tumor cells. Cancer Cell 4:463–476. CrossRefPubMedGoogle Scholar
  12. 12.
    Kitamura YI, Kitamura T, Kruse J-P et al (2005) FOXO1 protects against pancreatic β cell failure through NeuroD and MafA induction. Cell Metab 2:153–163. CrossRefPubMedGoogle Scholar
  13. 13.
    Zanella F, dos Santos NR, Link W (2013) Moving to the core: spatiotemporal analysis of forkhead box O (FOXO) and nuclear factor-κB (NF-κB) nuclear translocation. Traffic 14:247–258. CrossRefPubMedGoogle Scholar
  14. 14.
    Cautain B, Castillo F, Musso L et al (2016) Discovery of a novel, isothiazolonaphthoquinone-based small molecule activator of FOXO nuclear-cytoplasmic shuttling. PLoS One 11:e0167491CrossRefGoogle Scholar
  15. 15.
    Calnan DR, Brunet A (2008) The FOXO code. Oncogene 27:2276–2288. CrossRefPubMedGoogle Scholar
  16. 16.
    Wang R, Yang S, Nie T et al (2017) Transcription factors: potential cell death markers in parkinson’s disease. Neurosci Bull 33(5):552–560. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Cyert MS (2001) Regulation of nuclear localization during signaling. J Biol Chem 276:20805–20808. CrossRefPubMedGoogle Scholar
  18. 18.
    Martins R, Lithgow GJ, Link W (2016) Long live FOXO: unraveling the role of FOXO proteins in aging and longevity. Aging Cell 15:196–207. CrossRefGoogle Scholar
  19. 19.
    Terragni J, Graham JR, Adams KW et al (2008) Phosphatidylinositol 3-kinase signaling in proliferating cells maintains an anti-apoptotic transcriptional program mediated by inhibition of FOXO and non-canonical activation of NFκB transcription factors. BMC Cell Biol 9:6. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Van Der Heide LP, Hoekman MFM, Smidt MP (2004) The ins and outs of FOXO shuttling: mechanisms of FOXO translocation and transcriptional regulation. Biochem J 380:297–309. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Fundacion MEDINA Parque tecnologico ciencias de la saludGranadaSpain
  2. 2.Peptomyc S.L. CELLEX/Vall d’Hebron Institute of OncologyCarrer de Natzaret, 115Spain

Personalised recommendations