Fig. 6: Activation of ERK1/2 signaling pathway using a synthetic activator alleviates LH-induced adverse effects on endometrial stem cells.

A schematic diagram describing the role of the FAK/ERK1/2 signaling in controlling LH-mediated adverse effects on various endometrial stem cell functions is shown (A). Cells were prestimulated with 10 µM ERK1/2 phosphorylation activator ceramide C6 for 1 h before 25 nM LH treatments for 48 h. LH-induced adverse effects on the self-renewability of endometrial stem cells were evaluated using MTT-based assays (B). ERK1/2 activator ceramide C6 treatment alleviates LH-mediated inhibitory effects on migration potential as demonstrated using transwell assays (C) and western blotting for MMP-2 and MMP-9 (D). Cells were prestimulated with 10 µM ERK1/2 activator ceramide C6 for 1 h before 25 nM LH treatment for 48 h. ERK1/2 activator ceramide C6 treatment alleviates the LH-mediated inhibitory effects on adipocyte (E) and osteoblast F differentiation abilities as examined by oil red O and alizarin red S staining, respectively. ERK1/2 phosphorylation activator ceramide C6 (10 µM) alleviated the LH-mediated changes in the mRNA expression levels of multipotency-associated genes C-MYC, KLF4, NANOG, OCT4, and SOX2 as demonstrated using qPCR (G). β-actin was used as an internal control to normalize protein expression. PPIA was used as an internal control to normalize mRNA expression for qPCR analysis. All experiments were performed in triplicates. Data are presented as mean ± standard deviation (SD). *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t test).