Multiparameter analysis of apoptosis in puromycin-treated Saccharomyces cerevisiae
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ORIGINAL PAPER
Multiparameter analysis of apoptosis in puromycin‑treated Saccharomyces cerevisiae Barbara Citterio1 · Maria Cristina Albertini1 · Lina Ghibelli2 · Elisabetta Falcieri3 · Michela Battistelli3 · Barbara Canonico3 · Marco B. L. Rocchi1 · Laura Teodori4 · Maurizio Ciani5 · Elena Piatti1
Received: 5 November 2014 / Revised: 1 April 2015 / Accepted: 2 April 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract In Saccharomyces cerevisiae, a typical apoptotic phenotype is induced by some stress factors such as sugars, acetic acid, hydrogen peroxide, aspirin and age. Nevertheless, no data have been reported for apoptosis induced by puromycin, a damaging agent known to induce apoptosis in mammalian cells. We treated S. cerevisiae with puromycin to induce apoptosis and evaluated the percentage of dead cells by using Hoechst 33342 staining, transmission electron microscopy (TEM) and Annexin V flow cytometry (FC) analysis. Hoechst 33342 fluorescence images were processed to acquire parameters to use for multiparameter analysis [and perform a principal component analysis, (PCA)]. Cell viability was evaluated by Rhodamine 123 (Rh 123) and Acridine Orange microscope fluorescence staining. The results show puromycin-induced apoptosis in S. cerevisiae, and the PCA analysis indicated that the increasing percentage of apoptotic cells delineated a well-defined graph profile. The results were supported by TEM and FC. This study gives new insights into yeast apoptosis using puromycin as inducer agent, and PCA
Communicated by Olaf Kniemeyer. * Elena Piatti [email protected] 1
Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, Urbino, Italy
2
Department of Biology, University of Rome “Tor Vergata”, Rome, Italy
3
Department of Earth, Life and Environment Sciences, University of Urbino “Carlo Bo”, Urbino, Italy
4
UTAPRAD‑DIM, ENEA Frascati, Rome, Italy
5
Department of Life and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy
analysis may complement molecular analysis facilitating further studies to its detection. Keywords Saccharomyces cerevisiae · PCA · Apoptosis · Puromycin
Introduction Apoptosis is the term used to describe a type of cellular suicide that can occur in higher eukaryotes (Wyllie et al. 1980). Cell suicide responses have been documented in unicellular organisms including bacteria and eukaryotic cells (Jacobson et al. 1997; Engelberg-Kulka et al. 2004; Sharon et al. 2009; Carmona-Gutierrez et al. 2012). The findings, more than a decade ago, that baker’s yeast (Saccharomyces cerevisiae) can undergo apoptosis uncovered the possibility to investigate this mode of programmed cell death (PCD) in a model organism that combines both technical advantages and an eukaryotic ‘cell room.’ Since then, numerous exogenous and endogenous triggers have been found to induce yeast apoptosis, and multiple yeast orthologs of crucial metazoan apoptotic regulators have been identified and characterized at the molecular level (Engelberg-Kulka et al. 2004; Winderickx
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