Some are trophoblast stem cell associated genes, others are differentiation asso ciated genes, make it clear while still others were not affected by differentiation state. Genes listed in Additional file 5, Supplemental Table S5 are those with arbitrary expres sion signal strengths 800 in the differentiated cell con dition and displaying a significantly lower level of expression in the differentiated cell state versus the dif ferentiated cell state treated with the PI3K inhibitor. Of the 257 probe sets listed in Addi tional file 5, Supplemental Table S5, 99 genes were annotated by Ingenuity Pathway Analysis software. Functions associated with the annotated negatively regulated genes included cell survival, cellular assembly and organization, cellular growth and proliferation, cellular movement, and lipid metabolism.

These functions overlap with those observed for both the trophoblast stem cell associated and differentiation associated gene profiles. Of the sixteen validated trophoblast stem cell associated genes only Id2 was regulated by PI3K signaling. Klf2 and Rhob expression was not affected by differentiation state but was negatively regulated by PI3K. PI3K signaling, positively regulated genes The majority of positively regulated PI3K dependent genes are included in the differentiation associated gene set. Genes listed in Additional file 6, Supplemental Table S6 are those with arbitrary expression signal strengths 800 in the differentiated cell condition and displaying a significantly higher level of expression in the differentiated cell state versus the differentiated cell state treated with the PI3K inhibitor.

Of the 226 probe sets listed in Additional file 6, Supplemental Table S6, 90 genes were annotated by Ingenuity Pathway Analysis soft ware. Functions associated with the annotated positively regulated genes included cell survival, gene expres sion, cellular growth and proliferation, small molecule biochemistry, AV-951 cellular development, cellular movement, and lipid metabolism. These results are similar to that observed for the differentiation associated data set. Not all differentiation associated genes are regulated by PI3K suggesting that other signaling pathways contribute to the regulation of trophoblast differentiation. A subset of the positively regulated PI3K dependent genes identified from the microarray analysis was further evaluated by northern analysis or qRT PCR in Rcho 1 trophoblast cells treated with the PI3K inhibitor or vehicle. The differentiation associated genes sensitive to PI3K regulation have potential roles in cell invasion, immune and vascular cell regula tion, and the endocrine phenotype of tro phoblast giant cells.