Figure 1. BMP4 expression is elevated during Oleic acid-induced triglyceride/lipid accumulation in hepatocytes and in a mouse model of NAFLD. (A)BMP4 expression in Oleic acid-induced lipid accumulation. Primary mouse hepatocytes were stimulated with 0.05mM Oleic acid (methanol as a vehicle control). ORO staining was done at days 3, 5 and 7 respectively. Representative images are shown (a). Alternatively, total RNA was isolated at 36h and 72h post Oleic acid treatment and subjected to TqPCR analysis of Bmp4 expression. Relative expression was calculated by dividing the relative expression values (i.e., Bmp4/Gapdh) in “*” p < 0.05, Oleic acid group vs. methanol group (b). Total protein was isolated and subjected to Western blotting analysis of BMP4 expression at days 3, 5 and 7 post Oleic acid treatment (c). (B) Establishment of the mouse model of NAFLD. C57/B6 mice (4-week-old male, n=10 /time point/group) were fed with 45% high fat diet (HFD) or normal diet (Control), and sacrificed at weeks 10, 16 and 24, respectively. The retrieved liver tissue was subjected to H & E staining (a) and ORO staining (b). (C) BMP4 expression in mouse liver tissue of NAFLD. Total protein was isolated from the mouse liver tissue of the HFD and Control groups at weeks 10, 16 and 24 respectively, and subjected to Western blotting analysis of BMP4 expression. (a). IHC (immunohistochemical) staining of BMP4 expression was detected in the liver from the HFD and Control groups respectively (b). Each assay condition was done in triplicate, and representative images are shown or indicated by arrows.