Computational protocol: Programming of Obesity and Comorbidities in the Progeny: Lessons from a Model of Diet-Induced Obese Parents

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Protocol publication

[…] The total hepatic proteins of offspring were extracted in homogenizing buffer and protease inhibitors. Next, the homogenates were centrifuged for 20 min at 4°C and the supernatants were collected. Equal quantities of total protein were re-suspended in sodium dodecyl sulfate (SDS) containing sample buffer, heated for 5 min at 100°C, and separated by SDS-polyacrylamide gel electrophoresis. After electrophoresis, proteins were electroblotted onto a polyvinyl difluoride transfer membrane (Amersham Biosciences, Piscataway, NJ). In addition, the efficiency of the transfer was visualized by Ponceau solution staining. The membrane was blocked by incubation with nonfat dry milk (6% in Tween 20–Tris-buffered saline). Antibodies against sterol regulatory element binding protein-1c (SREBP-1c, 68 kDa), peroxisome proliferator-activated receptor alpha (PPAR-alpha, 55 kDa), fatty acid synthase (FAS, 270 kDa) and beta-actin (beta-actin, 43 kDa) were purchased from Santa Cruz (Santa Cruz Biotechnology, CA, USA). Following incubation with the primary antibody, the membrane was incubated with the secondary antibody for 1 h at room temperature. The membrane was developed using ECL Western blotting detection reagents, and the images of the blots were obtained with the Bio-Rad Molecular Imaging ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA). The intensity of the chemiluminescent bands was quantified using ImageJ software, version 1.49i (NIH,, USA). The blots were stripped and reprobed for beta-actin as a loading control to normalize the blot data. [...] Data are expressed as the mean and standard deviation and were tested for normality and homoscedasticity of the variances. The differences among groups were analyzed using one-way ANOVA followed by the posthoc test of Holm-Sidak (to test data between SC- and HF diet-fed mothers and fathers; and to study male and female offspring) (GraphPad Prism v. 6.05 for Windows, GraphPad Software, La Jolla, CA, USA). Interactions between paternal and maternal diets and offspring sex were evaluated through three-way ANOVA (2 x 2 x 2 factorial) (Statistica, v. 7; StatSoft Inc., Tulsa, OK, USA). The overall significance of a three-way effect and critical two-way interactions (paternal diet- offspring sex, maternal diet- offspring sex and paternal diet-maternal diet) were examined. In all cases, P<0.05 was considered statistically significant. […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Fatty Liver, Hyperglycemic Hyperosmolar Nonketotic Coma, Glucose Metabolism Disorders
Chemicals Cholesterol, Glucose, Triglycerides