Fig. 2

Acetylsalicylic acid exerts diverse effects on the regenerative capacity of endometrial stem cells through its target gene SERPINB2. A schematic representation of the regulatory role of SERPINB2 in mediating the diverse effects induced by acetylsalicylic acid is shown (A). Endometrial stem cells were treated with or without acetylsalicylic acid (2.5 mM) for 72 h. The in vitro effects of acetylsalicylic acid treatment on the mRNA and protein expression levels of SERPINB2 in a dose-dependent manner were analyzed using real-time PCR and western blotting, respectively (B). In addition, mice were intraperitoneally treated with acetylsalicylic acid (50 mg/kg daily for 7 consecutive days) or vehicle (PBS). Various stem cell types were isolated from the mouse uterine endometrium, bone marrow, and adipose tissues, and the changes in SERPINB2 expression were subsequently analyzed using real-time PCR and western blotting (C). Endometrial stem cells were transfected with specific shRNA targeting SERPINB2 and then treated with or without acetylsalicylic acid (2.5 mM); the subsequent changes in self-renewal capacity were analyzed at 48 h using MTT assays (D). The effects of SERPINB2 knockdown on the acetylsalicylic acid-induced changes in the migration potential of endometrial stem cells were also assessed by in vitro Transwell assays (E) and western blotting with antibodies against MMP-2 and MMP-9 (F). The effects of SERPINB2 depletion on the acetylsalicylic acid-induced inhibition of adipogenic (G) and osteogenic (H) differentiation were evaluated using oil red O staining and alizarin red S staining, respectively. The attenuating effects of SERPINB2 depletion on the acetylsalicylic acid-induced changes in the levels of the pluripotency/stemness-associated transcription factors NANOG, OCT4, and SOX2 were also assessed by real-time PCR (I). 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)