Fig. 6

Involvement of melatonin-activated Sp1/miR-597 pathway in BiP suppression. a UCB-MSCs were treated with melatonin (1 μM, 24 h) and total RNAs were extracted. MicroRNA microarray was conducted and the expression levels of microRNAs were analyzed using hierarchical clustering with heatmap. b A cluster of expression-increased microRNAs was selected and their expression levels under high cholesterol and melatonin condition were analyzed by qPCR (n = 5, *p < 0.05 vs control). c, d miR-597 mimic was transfected to UCB-MSCs for 24 h prior to cholesterol treatment (200 μM, 12 h). The mRNA expressions of HSPA5 and ABCA1 and protein expression levels of BiP and ABCA1 were quantified with qPCR and western blotting (n = 5, *p < 0.05 vs NT mimic, #p < 0.05 vs NT mimic + cholesterol). e, f MT2 inhibitor 4-P-PDOT (10 μM) was pretreated prior to melatonin treatment (1 μM, 12 h). e The phosphorylation of Sp1 (Thr453) were measured by western blotting (n = 5, *p < 0.05 vs control, ^#p < 0.05 vs melatonin). f Immunocytochemistry was conducted with Sp1 (green) specific antibody and DAPI (blue). Scale bar was set as 8 μm (magnification × 1000, n = 5, *p < 0.05 vs control, ^#p < 0.05 vs melatonin). g Sp1 inhibitor Mithramycin A (5 nM) was pretreated prior to melatonin treatment (1 μM, 12 h), and the change of expression level of miR-597-5p was assessed through qPCR (n = 5, *p < 0.05 vs control, #p < 0.05 vs melatonin). All images are representative and quantitative data are presented as a mean ± standard error of the mean