Fig. 6: Activation of the ERK1/2 signaling pathway attenuates glucocorticoid-induced inhibitory effects on various stem cell functions.

A schematic diagram regarding the functional role of the FAK/ERK1/2 signaling pathway in mediating glucocorticoid-induced effects is shown (A). Endometrial stem cells were pretreated with the ERK1/2 activator ceramide C6 (10 µM) for 1 h prior to treatment with 500 nM glucocorticoid for 48 h, and the subsequent glucocorticoid-induced changes in cell proliferation were analyzed by MTT assays. The percentage (%) of proliferating stem cells was calculated relative to the number observed in the vehicle control (B). The attenuating effects of ERK1/2 activation on glucocorticoid-induced changes in stem cell migration were analyzed by Transwell assays (C) and western blotting for MMP-2 and MMP-9 (D), respectively. Endometrial stem cells were pretreated with the ERK1/2 activator ceramide C6 (10 µM) for 1 h prior to an additional 48 h treatment with 500 nM glucocorticoids, and subsequent glucocorticoid-induced changes in adipocyte and osteoblast differentiation were analyzed by oil red O and alizarin red staining, respectively. The relative quantification of secreted calcium deposition and lipid droplet (LD) formation within differentiated cells was estimated by analyzing the absorbance of the solubilized cells at 500 nm and 570 nm, respectively (E). The attenuating effects of the ERK1/2 activator ceramide C6 (10 µM) on glucocorticoid-induced expression of the pluripotency-related transcription factors KLF4, OCT4, and SOX2 were measured by real-time PCR (F). β-actin was used as an internal control. PPIA was used as a housekeeping gene for real-time PCR analysis. All experiments were performed in triplicates, and the data has been presented as mean ± standard deviation (SD). P-value under 0.05 was presented in figures.