Journal: Diabetes

Article Title: Mitochondrial-Targeted Catalase Protects Against High-Fat Diet-Induced Muscle Insulin Resistance by Decreasing Intramuscular Lipid Accumulation.

doi: 10.2337/db16-1334

Figure Lengend Snippet: Figure 2—The protection of diet-induced muscle insulin resistance in MCAT mice is associated with decreased intramuscular lipid accumu- lation. Body weight– and age-matched MCAT and WT mice were fed HFD for 6 weeks and fasted 6 h before basal tissue sampling. A–C: Intramuscular triglyceride, total and individual LcCoA species, and total and individual DAG in gastrocnemius skeletal muscle of MCAT and WT mice. D–F: PKCu membrane translocation, IRS-1 phosphorylation on Ser1101, and AKT phosphorylation on Ser473 in gastrocnemius skeletal muscle in MCAT and WT mice fed an HFD (n = 8). Data are mean 6 SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by Student t test. Cyto., cytosol; IP, immunoprecipitation; Memb., membrane; NS, not significant.

Article Snippet: The primary antibodies used in the current study were as follows: phospho-Akt (Ser473), total Akt, phosphorIRS-1 (Ser1101), and PTEN (Cell Signaling; Beverly, MA); protein kinase C-u (PKCu) and total IRS-1 (BD Biosciences); GAPDH (Santa Cruz Biotechnology, Dallas, TX); catalase (Abcam, Cambridge, MA); and b-tubulin (Millipore, Billerica, MA).

Techniques: Sampling, Membrane, Translocation Assay, Phospho-proteomics, Immunoprecipitation

Journal: Science signaling

Article Title: Integrating network reconstruction with mechanistic modeling to predict cancer therapies.

doi: 10.1126/scisignal.aae0535

Figure Lengend Snippet: Fig. 4. p70S6K-mediated feedback inhibition of IRS1 in SW480, HCT116, HKE3, and HT29 cells. (A) Effects of AKT inhi- bition on p70S6KT421/S424 and IRS1S636/S639 phosphorylation in SW480, HCT116, HKE3, and HT29 cells. Starved cells were treated with AKT inhibitor VIII (10 mM) for 1 hour and then stimulated with TGFa (100 nM) for 0, 10, and 60 min. Phospho-AKTS473, phospho-IRS1S636/S639, total IRS1, phospho-p70S6KT421/S424, and total p70S6K were measured by Western blotting. (B) Effects of p70S6K knockdown (KD) on IRS1 phosphorylation in SW480, HCT116, HKE3, and HT29 cells. Cells were transfected with siRNA against p70S6K. Twenty-four hours later, cells were serum- starved for 4 hours, treated with TGFa, and analyzed as above. Blots were quantified using ImageJ. In (B) and (C), the phosphorylated proteins were normalized to the respective total proteins, and the normalized levels were scaled between 0 and 1 and plotted. Error bars were calculated from n = 3 independent experiments (figs. S2 and S3). P values were calculated using Kruskal-Wallis test. (C) Graphical summary of the results. The squares and circles represent the positive (+Ve) or negative (−Ve) interaction strengths, respectively. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Membranes were blocked with tris-buffered saline (TBS)–Tween (pH 7.4) containing 5% skim milk for 1 hour and incubated with 1:1000 diluted rabbit anti-human phospho-AKT (Ser473), AKT, phospho-p44/42 MAPK (Thr202/Tyr204), p44/42 MAPK, p70 S6 kinase, phospho-p70 S6 kinase (Thr389), IRS1, and phospho-IRS1 (Ser636/Ser639) antibodies or as control with mouse anti-human GAPDH (all from Cell Signaling Technology Inc.) in TBS-Tween with 5% bovine serum albumin overnight at 4°C.

Techniques: Inhibition, Phospho-proteomics, Western Blot, Knockdown, Transfection

Journal: Science signaling

Article Title: Integrating network reconstruction with mechanistic modeling to predict cancer therapies.

doi: 10.1126/scisignal.aae0535

Figure Lengend Snippet: Fig. 7. The role of the p70S6K-mediated negative feedback to IRS1 in EGFR inhibitor resistance of HCT116 cells. (A) Simulated active ERK (blue) and AKT (red) concentra- tions after p70S6K knockdown and different levels of EGF or TGFa (EGF/TGFa) stimulations. Solid lines and shaded areas represent means and SDs. (B) Effects of p70S6K knockdown on AKTS473 and ERK1/2T202/Y204 phosphorylation. HCT116 cells were transfected with nontargeting siRNAs (CTRL) or siRNAs against p70S6K (KD) and grown in serum (see fig. S2 for knockdown efficiency) for 24 hours. Then, starved (4 hours) cells were stimulated with TGFa (100 nM) for the indicated time points. Protein levels were quantified and normalized as above. Data are representative of n = 3 independent experiments (fig. S6). P values were calculated using Kruskal-Wallis test. (C) Simulated effects of p70S6K knockdown and EGFR inhibition on AKT activation in serum-grown HCT116 cells. The starting amounts of ligand were set to 0.01 AU. (D and E) Effects of p70S6K knockdown and EGFR inhibition on AKTS473 phosphorylation in serum-grown HCT116 cells. Control and knockdown HCT116 cells were treated with the EGFR inhibitor BIBX1382 (BIBX; 5 mM). Phosphorylated AKTS473 levels were measured at the indicated time points after treatment and normalized by total AKT levels. Data are from n = 3 independent experiments (fig. S5). P values were calculated using Kruskal-Wallis test. A representative blot is shown in (E). (F) Simulation of AKT activity for different p70S6K knockdown efficiencies and EGFR inhibitor strengths. (G) Phosphorylated AKT S473 levels in control and knockdown HCT116 cells with and without BIBX1382 treatment. Data represent n = 3 independent experiments. P values were calculated using Kruskal-Wallis test. (H) Feedback loops that control AKT phosphorylation. Positive feedback loops (highlighted in blue) controlling AKT phosphorylation and negative feedback loops (highlighted in red) controlling AKT phosphorylation.

Article Snippet: Membranes were blocked with tris-buffered saline (TBS)–Tween (pH 7.4) containing 5% skim milk for 1 hour and incubated with 1:1000 diluted rabbit anti-human phospho-AKT (Ser473), AKT, phospho-p44/42 MAPK (Thr202/Tyr204), p44/42 MAPK, p70 S6 kinase, phospho-p70 S6 kinase (Thr389), IRS1, and phospho-IRS1 (Ser636/Ser639) antibodies or as control with mouse anti-human GAPDH (all from Cell Signaling Technology Inc.) in TBS-Tween with 5% bovine serum albumin overnight at 4°C.

Techniques: Knockdown, Phospho-proteomics, Transfection, Inhibition, Activation Assay, Control, Activity Assay

Journal: Cells

Article Title: Fibroblast Growth Factor 21 (FGF21) Administration Sex-Specifically Affects Blood Insulin Levels and Liver Steatosis in Obese A y Mice

doi: 10.3390/cells10123440

Figure Lengend Snippet: TaqMan Gene Expression Assays used for relative quantitation real-time PCR.

Article Snippet: Insulin receptor substrate 1 , Irs1 , Mm01278327_m1.

Techniques: Gene Expression, Quantitation Assay, Derivative Assay