Journal: Bioorganic & Medicinal Chemistry

Article Title: Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system

doi: 10.1016/j.bmc.2010.06.053

Figure Lengend Snippet: Figure 2. Intracellular analysis of repressor pairs simulating Hdm2–p53 and Hdmx–p53 interactions in the E. coli reporter strain. (A and B) Growth analysis through droplet inoculation of serially diluted strains (10–106 colony forming units (cfu)) expressing the Hdmx–p53 pair of DBD fusions (row 1), the Hmd2–p53 pair of DBD fusions (row 2), a constitutively active repressor control (row 3) (Ref. 22) and an unrepressed (DBDs only) control (row 4) on a (A) non-selective inducing medium (LB with 100 lM IPTG) and (B) selective inducing medium (LB with 100 lM IPTG and 25 lg/mL kanamycin). (C) Reporter-gene (b-galactosidase) activity analysis of Hdm2–p53 and Hdmx–p53 strains. The arrow indicates the expression level (32 lM) for achieving approximately 90% reduction in the reporter expression to be used in the selection procedure.

Article Snippet: Linear p53 peptide ETFSDLWKLL was synthesized by ChemImpex, Inc., in 95% HPLC purity.

Techniques: Expressing, Control, Activity Assay, Selection

Journal: Bioorganic & Medicinal Chemistry

Article Title: Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system

doi: 10.1016/j.bmc.2010.06.053

Figure Lengend Snippet: Figure 3. SDS–PAGE analysis of the affinity capture–elution assay performed with the selected SICLOPPS hits. The unspliced constructs containing sequences CIFYYV and CDLRWF were immobilized via chitin-binding domain (CBD) fusion fragments on chitin beads and incubated with an equimolar mixture of Hdm2 or Hdmx (1 mM). Retained materials were subsequently treated with a solution (1 mM) of a p53-derived peptide (ETFSDLWKLL), and the eluate as well as other components of the assay were analyzed by SDS–PAGE. The lane assignments are as follows: lane 1 is a protein ladder; lanes 2 and 3 correspond to the CIFYYV and CDLRWF SICLOPPS constructs, respectively, isolated by chitin beads from crude overexpression lysates; lanes 4 and 5 contain purified Hdm2 and Hdmx, respectively; lane 6 corresponds to an equimolar mixture of Hdm2 and Hdmx; lanes 7 and 8 contain protein material eluted with the p53-derived peptide from the affinity supports containing CIFYYV and CDLRWF leads, respectively, which were pretreated with the equimolar mixture of Hdm2 and Hdm; lanes 9 and 10 were loaded with the post-elution material retained by the chitin beads pre-functionalized with the CIFYYV and CDLRWF constructs, respectively.

Article Snippet: Linear p53 peptide ETFSDLWKLL was synthesized by ChemImpex, Inc., in 95% HPLC purity.

Techniques: SDS Page, Construct, Binding Assay, Incubation, Derivative Assay, Isolation, Over Expression

Journal: Bioorganic & Medicinal Chemistry

Article Title: Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system

doi: 10.1016/j.bmc.2010.06.053

Figure Lengend Snippet: Figure 4. Performance of the selected SICLOPPS constructs and the corresponding single-alanine mutants in the reporter-gene and growth rate assays. (A and B) ONPG assay data and droplet inoculation analysis, respectively, of the anti-Hdm2 CIFYYV construct and its mutants in the Hdm2–p53 strain. (C and D) ONPG assay data and droplet inoculation analysis, respectively, of CDLRWF and its mutants in the Hdmx–p53 strain.

Article Snippet: Linear p53 peptide ETFSDLWKLL was synthesized by ChemImpex, Inc., in 95% HPLC purity.

Techniques: Construct

Journal: Bioorganic & Medicinal Chemistry

Article Title: Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system

doi: 10.1016/j.bmc.2010.06.053

Figure Lengend Snippet: Figure 5. Surface representations of Hdm2 (PDB: 1YCR; left) and Hdmx (PDB: 3DAB; right) in bound states with ligands (p53 residues 15–29) not shown for clarity. The proteins are colored according to the elemental make-up (C, gray; O, red; N, blue; S, yellow). The p53-binding pockets with labeled Leu, Trp and Phe subsites (green) are outlined to highlight the topological differences in the respective binding pockets. The residues proposed to be responsible for differences in ligand recognition patterns (F86 and H96 in Hdm2; L85 and P95 in Hdmx) are indicated by arrows.

Article Snippet: Linear p53 peptide ETFSDLWKLL was synthesized by ChemImpex, Inc., in 95% HPLC purity.

Techniques: Binding Assay, Labeling

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: A Monolayers were stimulated with GLP or Exendin-4 for 5 min, 15 min, 30 min. (10 nM) as described in MATERIALS AND METHODS. Bars show % decrease in bioluminescence compared with unstimulated (no ligand) monolayer (means ± SE; *p < 0.05 compared with unstimulated monolayer). The experiment was repeated three times and compared with untreated controls and pre-immune serum treated controls. B: Cell fractionation of HuH7 monolayers was performed as described in MATERIALS AND METHODS. Samples from membrane (M), cytoplasm(C) and nuclear (N) fractions were subjected to Western blot analysis using anti-GLP-1R antibody (1:500). Blots were also probed for Na+-K+-ATPase, Lamin A/C and β-actin to confirm equal protein loading. The results are representative of 2 independent experiments. C: Confocal imaging of GLP-1R was performed on filter grown monolayers of HuH-7 cells. Cells were stained with rabbit polyclonal antibody against GLP-1R (1:200) followed by Alexa Fluor secondary antibody. Rhodamine (blue arrow) was used to stain the cytoskeleton. In 1) GLP-1R (yellow arrow) is seen localized to the membrane and in 2) upon agonist stimulation GLP-1R is decreased from the membrane.

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: Cell Fractionation, Western Blot, Imaging, Staining

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: A HuH7 cells were treated with palmitic acid ( 400uM/l) and oleic acid ( 400uM/l) for 12 h under insulin-free conditions; and subsequently exposed to Exendin-4 (20nM) for 6 h. Figure 3a) shows Oil red O staining of HuH7 cells treated with FFA and Exendin-4. A marked increase in Oil red O stained droplets (red) are visible in the cells treated with FFA as compared with the non treated cells. On exposure to Exendin4 there is a significant loss of fat droplets (40X). B: Triglyceride assay was performed on HuH7 cell lysate after treatment with palmitic and oleic acid followed by exposure to Exendin-4 as described in MATERIALS AND METHODS. Bars show % increase in TG content and then % decrease on treatment with Exendin-4. (Means ± SE; *p < 0.05 compared with untreated steatotic cells). The experiment was repeated three times in triplicate and compared with FFA exposed and non Exendin4 treated controls. C.: HepG2 cells were grown in either control media or methionine-choline deficient (MCD) media. Cells were then treated with Exendin-4 for 24 h. Following treatment, intracellular lipids (polar and neutral) were stained with Nile Red (NR) (0.5μg/ml). Flow cytometry was performed as described in Materials and Methods. 3T3L1 cells were served as a positive control. The figure is representative of three independent experiments.

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: Staining, Flow Cytometry, Positive Control

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: HuH-7 cells were treated with GLP/Exendin-4 (10nM) following the time course indicated: 5, 15, 30, 60, 90 and 120 m and Western blot was performed. β-actin was used as loading control. A: Phosphorylation of PDK was induced. B–C: AKT phosphorylation was also increased in a time dependent manner as was the phosphorylation of PKC-ζ. All data presented are representative of the mean ± SE of at least 3 experiments *p<0.05 vs. basal or untreated.

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: Western Blot

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: HuH7 cells were transfected with siRNA (at 30nM) against GlP-1R, and Western blot analysis with β-actin serving as loading control was performed. A) Knockdown was achieved as compared to control with 30nM siGLP-1R. B) Transfected HuH7 cells were treated with Exendin-4 (10nM) for 60 min. siRNA GLP-1R abolished the Exendin-4 mediated-effects on PDK-1 and PKC-ζ. (B and C, respectively). These studies represent multiple independent experiments. (*p<.05 vs. control).

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: Transfection, Western Blot

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: In our previous work we demonstrated that GLP-1 or Exendin-4 increased cAMP production. Here we propose that the GLP-1 action shares key downstream components of the insulin signaling pathway, including PKCζ, which has been shown to be a key factor in NAFLD.

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: