Fig. 3.

Changes in the steatosis phenotype upon CYP4A overexpression or knockdown in a novel 3D hepatic steatosis model. HepaRG cells were transduced with retroviral CYP4A (RV-CYP4A) or transfected with an siRNA for CYP4A (siCYP4A) and then 3D cultured with HUVECs and MSCs for 10 days. Steatosis was induced with glucose/palmitate for the last 5 days. a mRNA expression levels of CYP4A and b CYP4A activity were determined in each indicated 3D spheroid; n = 3. c mRNA expression levels of gluconeogenesis- and lipogenesis-related genes were determined in each 3D spheroid; n = 3. β-Actin was used as an internal control. d Protein expression levels of CYP4A and gluconeogenesis- and lipogenesis-related genes were determined in each group. e Intracellular lipids were stained with Nile red (left) and quantified by fluorescence intensity (right); n = 3. f The triglyceride concentration was quantified by absorbance in each treated 3D spheroid; n = 3. g Glucose uptake was quantified by fluorescence intensity; n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001