Fig. 7. Knocking down ACER3 elevates C_18:1-ceramide and protects human hepatocytes from apoptosis and oxidative stress in response to overload of palmitate.

a and b Human L02 hepatocytes were transfected with each of two short hairpin RNAs (shRNA) specific for the ACER3 gene (shACER3-1 and shACER3-2) or a control shRNA (shCON), ACER3 mRNA a and enzymatic activity b were measured by qPCR and in vitro activity assays, respectively. c L02 cells transfected with shACER3-1, shACER3-2, or shCON were treated with BSA or BSA–palmitate complex (100 μM) for 6 h before the levels of ceramides were determined by LC–MS/MS. d and e L02 cells transfected with shACER3-1, shACER3-2, or shCON were treated with BSA or BSA–palmitate complex (100 μM) for 24 h before ORO staining d or MTT assays were performed e. The optical density of ORO staining was measured as described in “Materials and methods” section. f L02 cells transfected with shACER3-1, shACER3-2, or shCON were treated with BSA or BSA–palmitate complex (100 μM) for indicated time durations before western blot analyses were performed with an antibody against PARP, cleaved caspase 3, or β-Actin (a protein-loading control). g L02 cells transfected with shACER3-1, shACER3-2, or shCON were treated with BSA or BSA–palmitate complex (100 μM) for 12 h before Western blot analyses were performed with the antibody against 4-HNE or β-actin (a protein-loading control). h and i L02 cells transfected with shACER3-1, shACER3-2, or shCON were treated with BSA or BSA–palmitate complex (100 μM) for 12 h before DHE staining was imaged h with background in white and the staining in black, and the florescent intensity was detected as described in “Materials and methods” section i. Data in a–e and i represent results mean ± SD of three independent experiments. Images in f–h represent results from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.