Fig. 4

Activation of the Akt signaling cascade alleviates the acetylsalicylic acid-induced suppression of various endometrial stem cell functions related to regenerative capacity. A schematic representation of the regulatory role of the PI3K/Akt signaling cascade in mediating the effects of acetylsalicylic acid is shown (A). Endometrial stem cells were pretreated with the specific Akt activator SC79 (10 µM) for 1 h and then treated with acetylsalicylic acid (2.5 mM) for 48 h, and the subsequent effects of acetylsalicylic acid on self-renewal capacity were assessed using MTT assays. The stem cell proliferation rates (%) were assessed by representing the viability of the acetylsalicylic acid-treated cells as a percentage of the viability of the vehicle-treated cells (B). The effect of the Akt activator on the acetylsalicylic acid-induced change in migration potential was evaluated by in vitro Transwell assays (C) and western blotting with antibodies against MMP-2 and MMP-9 (D). Endometrial stem cells were pretreated with 10 µM SC79 for 1 h and then treated with 2.5 mM acetylsalicylic acid for 48, and the subsequent changes in adipogenic (E) and osteogenic (F) differentiation were analyzed by oil red O and alizarin red staining, respectively. The cytoplasmic calcium concentration and lipid droplet (LD) formation within differentiated cells were assessed by measuring the absorbance values of the solubilized cells at wavelengths of 500 nm and 570 nm, respectively. The effect of 10 µM SC79 on the acetylsalicylic acid-induced inhibition of the pluripotency-associated transcription factors NANOG, OCT4, and SOX2 was measured by real-time PCR (G). β-actin was used as an internal control to normalize protein expression. PPIA was used as a reference gene to normalize gene expression. All experiments were performed in triplicates. Significant differences are presented. *p < 0.05, **p < 0.005, and ***p < 0.001 (One-way ANOVA)