Figure 5

Klf10 deficiency aggravates liver injury during steatohepatitis in mice and hepatocyte death in vitro. (A) Serum ALT activity of WT and Klf10 ^-/- male mice were fed a methionine and choline deficient diet (MCDD) for 4 weeks and sacrificed at ZT 3 and 21 (n = 8–20 mice/group). (B–D) Mouse primary hepatocytes were transfected with control (siCtrl) or Klf10 (siKlf10) siRNA and were stimulated with TNFα (20 ng/mL) and actinomycin D (0.1 µg/mL) for 16 h (B–C) or 12 h (D). (B) Hepatocyte Klf10 gene expression (n = 6). Gene expression is normalized to B2m and expressed as relative expression of the untreated siCtrl condition, 72 h after transfection. Hepatocytes cell viability assessed by MTT assay (n = 6). (C) Hepatocyte cell death and apoptosis assessed by flow cytometry following 7aaD and annexinV staining (n = 5). (D) Western blot of cleaved CASPASE 3 fragment (17 kDa) (c-CASP3) and HSP90 protein (left) and densitometry quantification of cleaved CASPASE 3 was normalized to HSP90 in four independent experiments (right). All the data are expressed as mean ± SEM. Statistical significance was tested using the Mann–Whitney test; * and $ p < 0.05, ** and $$ p < 0.01,*** and $$$ p < 0.001, vs control group (A)/cells (B,C,D) (stars) or the indicated groups (A)/cells (B, C, D) (dollar symbols).