Peroxidase activity was demonstrated using the ImmPact DAB (diaminobenzidine-based) peroxidise substrate kit (Vector Laboratories). Slides were counterstained with hematoxylin. Data are expressed as mean ± standard error of the mean (SEM), unless otherwise stated. Multiple comparisons on data sets were performed using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test. P ≤ 0.05 was regarded as significant. Influence of maternal obesity and postnatal diet as well as any interaction between them were investigated

using two-way ANOVA (GraphPad Prism 5.0; GraphPad Software, Inc., Cary, NC). The statistical unit was considered the number of dams (n = 5 per group), with one offspring

studied per litter per time point. Ab, antibody; α-SMA, alpha smooth muscle actin; ALT, alanine aminotransferase; ANOVA, analysis of variance; Col1-α2, collagen type 1 Vincristine order alpha 2; FACS, fluorescence-activated cell sorting; FCA, flow cytometric analysis; HBSS, Hank’s balanced salt solution; H&E, hematoxylin and eosin; Ig, immunoglobulin; IHC, immunohistochemical; IL, interleukin; IR, insulin resistance; KCs, Kupffer cells; LPS, lipopolysaccharide; mAb, monoclonal Ab; MNC, mononuclear cell; mRNA, messenger RNA; NAFLD, nonalcoholic fatty liver disease; Seliciclib mw NAS, NAFLD Activity Score; NASH, nonalcoholic steatohepatitis; NKT, natural killer T cells; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; ROS, reactive oxygen species; SEM, standard error of the mean; TGs, triglycerides; TGF-β, transforming growth factor beta; Th-1/2, T-helper cells; TNF-α, tumor necrosis factor alpha Both maternal

obesity and postnatal diet were independent predictors of offspring body weight, inguinal fat mass, and hepatic TG, and there was an apparent interaction between maternal and postnatal environments. Body weight (Fig. 1A) was significantly increased in OffCon-OD, compared to OffCon-SC. Body weight was further increased by exposure to maternal obesity during gestation and lactation. A similar pattern was observed for inguinal fat pad mass (Fig. 1B) and for hepatic TG content (Fig. 1C). Therefore, the postnatal high-fat diet induced increases in body weight, adiposity, and hepatic fat content and was exacerbated by previous exposure to maternal obesity. There was an independent MYO10 effect of both maternal obesity and postnatal high-fat diet on TNF-α, TGF-β and Col1-α2 expression as well as a significant interaction apparent between maternal obesity and the postnatal diet for TGF-β and Col1-α2. IL-6 and TNF-α, as markers of liver injury, were significantly up-regulated in OffCon-OD (Fig. 2A,B) and further significantly increased in OffOb-OD. Similarly, relative expression of α-SMA, TGF-β, and Col1-α2, as markers of hepatic fibrogenesis (Fig. 2C-E), were up-regulated in OffCon-OD and OffOb-OD, compared to OffCon-SC.