Fig 1.
(A) Treatment with cAMP (0.5 mM) and MPA (10-6M) for 9 days induced a morphological change in HIESC from a spindle to an ovoid shape. Images were taken using an Olympus BX60 microscope at 40x magnification. (B) mRNA were analysed by RT-PCR. β-actin was used as an internal control. Image shown are from one representative experiment. Graphics represent densitometric analysis. (C) Induction of PRL secretion by the same treatment in HIESC at different days of culture (1, 3, 6 and 9). A significant increase was found at days 6 and 10 with maximal levels observed at day 6 (P<0.0001). Expression of decidual marker genes, IGFBP1 (D) mRNA expression of either PRL or IGFBP1 were quantified following three day treatments. 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, RNA was extracted and qRT-PCR analyses were performed to quantify PRL or IGBP1 expression. 18S mRNA expression was used as control for qPCR results. All data are means ± SEM three independent experiments. Different letters represent significantly different means (p<0.05).
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.
Fig 3.
In vitro modulation of PI3K/Akt pathway with the induction of decidualization.
Induction of decidualization was induced with cAMP (0.5 mM) and MPA (10μM) for 48h, then MG132 was added and incubated for another 24h. (A) Treatment using Mg132 increased total ubiquitination, both in control conditions and decidualized cells, indicating that the Mg132 was effective at inhibiting proteasomal degradation. (B) Total Akt and pAkt levels were measured by Western blot. (C) Individual Akt isoforms levels were assessed by Western blot. β-actin was used as loading control. Blots shown are from one representative experiment. Graphics represent Western blot densitometric analysis. (D) RT-PCR was performed for each Akt isoforms to evaluate change in mRNA transcription. Data represent means ± SEM for three independent experiments. β-actin was used as an internal control. Image shown are from one representative experiment. Graphics represent densitometric analysis. All data are means ± SEM three independent experiments. Different letters represent significantly different means (p<0.05).
Fig 4.
In vitro modulation of PI3K/Akt pathway and their effects with the 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. mTOR and pmTOR protein expression (A), Slug protein expression (B) and IκB/pIκB protein expression (C) and were measured during decidualization. Total proteins were collected on different days of decidualization. β-actin was used as loading control. Blots shown are from one representative experiment. (D) Cells were harvested after three days of cAMP (0.5 mM) and MPA (10μM) treatment; they were then counted; an equal amount of cells were lysed and subsequently loaded to perform Western blot analysis. Changes in total IκB and phosphorylated IκB was quantified. (E) 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 IκB and p-IκB; β-actin was used as loading control. Presented Western blots 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.
Fig 5.
Subcellular localization of proteins following decidualization.
Decidualization was induced with cAMP (0.5 mM) and MPA (10μM) for three days (A) Cells were then harvested and cytoplasmic/nuclear extraction was performed following manufacturer protocol. Western blot was performed to qualify the change in Par-4, p65 and FoxO1. PARP was used as nuclear fraction control while GAPDH was used as cytoplasmic fraction control. The presented blot is of one representative experiment. (B) HIESC were seeded in 6-wells containing coverslips and grown in presence of cAMP (0.5 mM) and MPA (10μM) for three days. p65 and Par-4 localization were then observed by confocal microscopy. Presented images are of one representative experiment.
Fig 6.
Decidualization and inhibition of PI3K/Akt pathway reduces cell motility.
(A) Wound healing assays were performed using HIESC treated with either vehicle or cAMP (0.5 mM) and MPA (10μM). HIESC were allowed to grow until they reached confluence; cells monolayers were then scratched with the blunt end of a tip and images were captured at 0, 6, 12 and 24h postwounding in order to assess cell motility. Wound closure was quantified as the percentage of recovered area. (B) Wound healing assays were performed using HIESC treated with either vehicle or cAMP (0.5 mM) and MPA (10μM). HIESC were allowed to grow until they reached confluence; cells monolayers were then scratched with the blunt end of a tip and images were captured at 0, 6, 12 and 24h postwounding in order to assess cell motility. Treatment consisted of either vehicle (control), cAMP (0.5 mM) and MPA (10μM), 10μM Wortmannin (PI3K inhibitor) or 100 nM Rapamycin (mTOR pathway inhibitor) or combinations of these compounds. Wound closure was quantified as the percentage of recovered area. Data are means ± SEM three independent experiments. Different letters represent significantly different means (p<0.05). Images were taken using an Olympus BX60 microscope at 40x magnification.
Fig 7.
Effect of forced expression of Akt isoforms (CA-Akt) on PRL and IGFBP1 expression.
mRNA expression of either PRL (A) IGFBP1 (B) were quantified following three days treatments. HIESC cells transfected with either Akt1, Akt2 or Akt3 Tet-On vectors were subjected to decidualization using cAMP (0.5 mM) and MPA (10μM). They were then either concomitantly treated with doxycycline (1μg/mL) in order to induce the expression of the constitutive Akt isoform construct (cAMP+MPA+Doxycycline) or cells were allowed to decidualize for 24h before doxycycline was added (cAMP+MPA+Doxycycline 24H). Cells were lysed after three days and qRT-PCR analyses were performed to quantify PRL or IGBP1 expression. β-actin mRNA expression was used as control for qPCR results. (C) Akt 1, Akt2 and Akt3 expression was quantified following three days treatments. HIESC cells transfected with either Akt1, Akt2 or Akt3 Tet-On vectors were subjected to decidualization using cAMP (0.5 mM) and MPA (10μM). They were then either concomitantly treated with doxycycline (1μg/mL) in order to induce the expression of the constitutive Akt isoform construct (cAMP+MPA+Doxycycline). Cells were lysed after three days and qRT-PCR analyses were performed to quantify Akt1, Akt2 or Akt3 expression. β-actin mRNA expression was used as control for qPCR results. Data are means ± SEM three independent experiments. Different letters represent significantly different means (p<0.05).