Regulation of the PI3K/Akt pathway during decidualization of endometrial stromal cells
Fig 2
Expression of Akt isoform, total Akt and pAkt during induction of decidualization.
Cells were incubated in the presence or absence of cAMP (0.5 mM) and MPA (10μM) for a total of nine days. Total protein and RNA were then extracted. (A) Western blot was performed to quantify the change in specific Akt isoforms levels as well as total Akt levels. pAkt(ser473) was used to assess Akt activation β-actin was used as loading control. (B) Cells were counted after three days in either control media or under cAMP (0.5 mM) and MPA (10μM) treatment to assess the effect of decidualization on proliferation. Trypan blue exclusion dye was used to assess the number of dead cells in the samples. (C) Cells were incubated in the presence of cAMP (0.5 mM) and MPA (10μM) treatment for three days; they were then counted; an equal amount of cells were lysed and subsequently loaded to perform Western blot analysis. Changes in total Akt, specific Akt isoforms as well as phosphorylated Akt (ser473) was quantified. (D) Cells were incubated in the presence of either cAMP (0.5 mM), MPA (10μM) or a combination of both. Cells were lysed after three days and total proteins were extracted. Western blot was performed to quantify the change in total Akt, pAkt(ser473), Par-4 or FoxO1; β-actin was used as loading control. All blots shown are from one representative experiment. All graphics represent Western blot densitometric analysis. All data are means ± SEM three independent experiments. Different letters represent significantly different means (p<0.05); *, p<0.05; **, p<0.01; ***, p<0.001.