title: Suppression of the PI3K subunit p85α delays embryoid body development and inhibits cell adhesion.
abstract: Phosphatidylinositol-3-kinases (PI3Ks) exert a variety of signaling functions in eukaryotes. We suppressed the PI3K regulatory subunit p85α using a small interfering RNA (Pik3r1 siRNA) and examined the effects on embryoid body (EB) development in hanging drop culture. We observed a 150% increase in the volume of the treated EBs within 24 h, compared to the negative controls. Fluorescence Activated Cell Sorting (FACS) assays showed that this increase in volume is not due to increased cellular proliferation. Instead, the increase in volume appears to be due to reduced cellular aggregation and adherence. This is further shown by our observation that 40% of treated EBs form twin instead of single EBs, and that they have a significantly reduced ability to adhere to culture dishes when plated. A time course over the first 96 h reveals that the impaired adherence is transient and explained by an initial 12-hour delay in EB development. Quantitative PCR expression analysis suggests that the adhesion molecule integrin-β1 (ITGB1) is transiently downregulated by the p85α suppression. In conclusion we found that suppressing p85α leads to a delay in forming compact EBs, accompanied by a transient inability of the EBs to undergo normal cell-cell and cell-substrate adhesion.
authors.affiliation: Institute of Continuing Education, University of Cambridge, Madingley CB23 8AQ, UK. smrw2@cam.ac.uk
authors.forename: Susan M R
authors.initials: SM
authors.lastname: Gurney
authors.forename: Peter
authors.initials: P
authors.lastname: Forster
authors.forename: Ursula
authors.initials: U
authors.lastname: Just
authors.forename: Ralf
authors.initials: R
authors.lastname: Schwanbeck
mesh_terms: D015153:Blotting, Western; D002448:Cell Adhesion; D002454:Cell Differentiation; D058732:Embryoid Bodies / Q000166:cytology*; D055785:Gene Knockdown Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000235:genetics; D000081082:Phosphoinositide-3 Kinase Inhibitors; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering
chemical_list: D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D034741:RNA, Small Interfering