The p85 Regulatory Subunit of PI3K Mediates cAMP-PKA and Insulin Biological Effects on MCF-7 Cell Growth and Motility
Figure 4
Effects of S83 phosphorylation on MCF-7 cells proliferation and viability. (a) MCF-7 cells transiently transfected with p85WT, p85A, or p85D or with the empty vector were treated with 10 nM insulin or with 0.5% FCS. For the colorimetric MTT assay (A) cells were counted after 0, 24, 48, 72, and 96 hours ( compared to the control; compared to p85WT or p85D). (b) Cell cycle analysis was performed by fluorocytometric absorbent cell sorter (FACS) to determine the percentage of cells in G2 M, S and G1-phase ( compared to empty vector or p85WT or p85D). The data are the mean of three independent experiments performed in triplicate (). (c) For the clonogenic assay transfected cells were seeded into 6 multiwell plates in the presence of 10 nM insulin. Twenty-four hours later insulin was removed or lowered to 5 nM. Fifteen days later the clones were counted and their cellularity was evaluated by contrast microscopy. Clones were counted after 30′ fixing with a mixture of 6% glutaraldehyde and 0.5% crystal violet. The histogram represents the average number of colonies of four separate experiments performed in duplicate (). ( versus empty vector).
