Fig. 1

Acetylsalicylic acid treatment markedly suppresses multiple beneficial functions of endometrial stem cells in vitro. We evaluated whether acetylsalicylic acid treatment inhibits various regenerative capacity-associated functions of endometrial stem cells, such as self-renewal, migration, pluripotency and multilineage differentiation, in vitro (A). The inhibition of self-renewal capacity by treatment with multiple concentrations of acetylsalicylic acid (100 nM, 500 nM, 1 mM, 2.5 mM, and 5 mM) was analyzed at 48 h 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). Endometrial stem cells were treated with acetylsalicylic acid (2.5 mM) for 72 h, and the acetylsalicylic acid-induced inhibition of migratory potential was then analyzed by Transwell migration/invasion assays (C). The levels of the well-known migration regulatory proteins MMP-2 (72 kDa) and MMP-9 (92 kDa) in cells with or without acetylsalicylic acid treatment were analyzed using western blotting (D). Endometrial stem cells were incubated in adipogenic or osteogenic differentiation medium for 2 weeks with or without acetylsalicylic acid (2.5 mM) treatment. The acetylsalicylic acid-induced inhibition of the adipogenesis (E) and osteogenesis (F) of endometrial stem cells was analyzed using oil red O and alizarin red S 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 (E). The acetylsalicylic acid-induced inhibition of the expression of various pluripotency/stemness markers (C-MYC, NANOG, OCT4, and SOX2) was evaluated using 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)