Lipid loading induced EMT in HK-2 cells. HK-2 cells were made quiescent by serum-free medium for 24 hours and then maintained in serum-free medium (control) or serum-free medium containing 30 μg/ml cholesterol together with 1 μg/ml 25-hydroxycholesterol (lipid) for 24 hours. (A) Morphological changes of HK-2 cells under phase contrast microscopy (I and II, Scale bar: 50 μm) and immunofluorescence analysis of E-cadherin (III and IV, Scale bar: 25 μm) and α-SMA (V and VI, Scale bar: 25 μm) expression in cells with or without lipid treatment. (B) Real-time PCR for E-cadherin and α-SMA mRNA expression in HK-2 cells with or without lipid treatment. β-actin was used as mRNA loading control. (C and D) Western blot analysis for E-cadherin and α-SMA protein expression. The histogram shows the average volume density corrected by housekeeping control, β-actin. Data are expressed as mean ± SD. *P < 0.05 vs. control. All of the data shown in these studies are representative of at least three separate experiments.