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The Scientific World Journal
Volume 2014, Article ID 565839, 11 pages
Research Article

The p85 Regulatory Subunit of PI3K Mediates cAMP-PKA and Insulin Biological Effects on MCF-7 Cell Growth and Motility

1Department of Medicine and Health Sciences, University of Molise, Campobasso, Italy
2Department of Biology, University Federico II of Naples, Naples, Italy
3Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Italy
4Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy
5Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, Temple University, Philadelphia, PA, USA

Received 18 April 2014; Revised 17 June 2014; Accepted 18 June 2014; Published 9 July 2014

Academic Editor: Elisabetta Baldi

Copyright © 2014 E. Di Zazzo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Supplementary Mathods.

Transfection efficiency determined by FACS analysis.

For the immunofluorescence experiment, the MCF7 cells were transiently transfected with p85WT and its mutants, as followed described: 2,5”106 cells were seeded in 100mm plates containing 9 round coverslips (GG-12-gelatin neuVitr0). After 12h, cells were transfected as described in Section 2 and 48h later, the coverslips were picked up and stained for immufluorescence as described in Section2. The remnant cells were harvested and fixed with paraformaldehyde (2% w/v in PBS) for 10 min and ethanol 70% for 20 min; permeabilized with Triton X-100 (0,2% v/v in PBS) for 20 min. Then cells were stained with anti-FLAG antibody diluited 111000 and with Alexa-Fluor 488 anti-mouse 111000 in PBS, and analysed by FACS for the transfection efficiency (see Figure S3B). For all others experiments the MCF7 cells were transiently transfected with p85WT or its mutants in the presence of pEGFPC3 plasmid. After 48h, the cells were harvested and analyzed for transfection efficiency by FACS analysis.

  1. Supplementary Material