Fig. 3. GV101 and KD025 inhibit adipogenesis and fibrogenic pathways.

Human subcutaneous preadipocytes were induced adipogenesis with a medium containing differentiation cocktail (DM) for 7 days in the presence of indicated inhibitors. Differentiated cells were stained at day 8 by Oil Red O (a), quantification of lipid accumulation was measured by absorbance at 492 nM of extracted Oil Red O, all the absorbance was normalized to vehicle (DMSO/DM) treated differentiated cells (a), total RNA was extracted at day 8 and Glut4 gene expression was accessed by real-time RT-PCR (b). Human subcutaneous preadipocytes were treated with indicated inhibitors for 2 h before whole cell extracts were collected for Western blot analysis of phosphorylated AMPK and β-Actin (c). MRC-5 cells were pre-treated with various concentration of indicated inhibitors for 1 h before TGF-β1 stimulation for 48 h. Whole cell extracts were subjected to Western blot for the expression of α-SMA, pCofilin and β-actin (d). Western blots were quantified and normalized to the β-actin, and values are indicated under the corresponding immunoblots. Col1α levels in supernatants were measured by ELISA. All the Col1α levels were normalized with that of vehicle (DMSO/TGF-β1) treated cells (d), gene expression of α-SMA, CTGF, Col3α levels were measured by real-time RT-PCR. All the levels were first normalized with internal 18s control and then were normalized with the level of vehicle (DMSO/TGF-β1) treated cells (e). The data (a, d) are representative of three repeated experiments, (b, e) are average of three repeated experiments.