Fig. 5

Activation of the ERK1/2 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 FAK/ERK1/2 signaling cascade in mediating the effects of acetylsalicylic acid is shown (A). Endometrial stem cells were pretreated with the specific ERK1/2 activator ceramide C6 (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 by 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 ERK1/2 activator on the acetylsalicylic acid-induced changes in migratory capacity 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 ERK1/2 activator ceramide C6 for 1 h and then treated with 2.5 mM treatment for 48, and the subsequent effects of acetylsalicylic acid on adipogenic (E) and osteogenic (F) differentiation were evaluated by oil red O and alizarin red staining. 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 ceramide C6 on the acetylsalicylic acid -induced inhibition of the pluripotency-associated transcription factors NANOG, OCT4, and SOX2 was analyzed 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)