Bah66 0100

Palmitic acid (PA; P0500; Sigma-Aldrich) and oleic acid (OA; O-1008; Sigma-Aldrich) stimulation was used to construct hepatocyte NAFLD model in vitro as previously reported (22 (link)). Fatty acid-free BSA (0.5%; BAH66-0100; Equitech Bio, Kerrville, TX) was used as a control. For Nile red staining, cells were fixed with 4% paraformaldehyde and stained with Nile red (catalog no.: 22190; 1 μM in PBS; Fanbo Biochemicals Co, Ltd, Beijing). Images were acquired with a laser scanning confocal microscope (TCS SP8; Leica, Wetzler, Germany).

Human hepatocyte L02, human embryonic kidney 293, and 293T cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All the cell lines were examined for mycoplasma contamination, and the results were negative. The cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin–streptomycin in a 5% carbon dioxide/water-saturated incubator at 37 °C. Before the experiments, all cell lines were verified through short tandem repeat DNA profiling. All the cell lines in our laboratory were passaged no more than 30 times after resuscitation and routinely tested for mycoplasma contamination by PCR. To establish a hepatic steatosis model in vitro, mouse primary hepatocytes and L02 cells were stimulated with palmitic acid (PA; 0.5 mM; P0500; Sigma-Aldrich; St. Louis, MO, USA) and oleic acid (OA; 1.0 mM; O-1008; Sigma-Aldrich; St. Louis, MO, USA) (dissolved in 0.5% fatty acid-free BSA) at the stated concentrations for 12–24 h. For the control group, cells were stimulated with fatty acid-free BSA (0.5%; BAH66-0100; Equitech Bio, Kerrville, TX, USA). To determine whether LAPTM5 facilitates the proteasomal degradation of CDC42, the cells were treated with 50 μmol/L of Chlq (S6999; Selleck Chemicals) for 12 h.