Journal: Journal of cell science

Article Title: Aminopeptidase P3, a new member of the TNF-TNFR2 signaling complex, induces phosphorylation of JNK1 and JNK2.

doi: 10.1242/jcs.149385

Figure Lengend Snippet: Fig. 6. Phosphorylation of JNK and IkB is induced by APP3 during TNFR1 or TNFR2 signaling. (A) HEK293T-TNFR2 cells transfected with APP3m or APP3c, or APP3 shRNA, were prepared. To inhibit aminopeptidase P enzymatic activity, HEK293T-TNFR2 cells were pre-treated with 100 mM apstatin. Untransfected cells were used as a reference. These cells were stimulated with R2-7 (100 ng/ml) for the indicated time. After cell lysis, western blotting was performed to confirm expression of APP3 and TNFR2, as well as phosphorylation of JNK (P-JNK) JNK1 and JNK2 are indicated by the open arrow and filled arrow, respectively. b-actin served as a loading control. (B) IkB and phospho-IkB were also detected in each HEK293T-TNFR2 cell line by western blotting. (C) HEK293T cells transfected with APP3m or APP3c, or APP3 shRNA, and pre-treated with 100 mM apstatin were stimulated by TNF (100 ng/ml) for the indicated time. After cell lysis, JNK and phospho-JNK (JNK1, open arrow; JNK2, filled arrow) were detected by western blotting. (D) IkB and phospho-IkB were also detected in each HEK293T cell line.

Article Snippet: Two APP3 shRNA plasmids, each with a different target sequence (APP3 shRNA A, 59-AGACCAGACAGTGGTTGTGCTCTCCAACC39; and B, 59-GATGGAGGTTGTGAGTCTTCCTGCTATGT-39), and a scrambled negative control shRNA plasmid (OriGene) were transfected into the cells by lipofection.

Techniques: Phospho-proteomics, Transfection, shRNA, Activity Assay, Lysis, Western Blot, Expressing, Control

Journal: Journal of cell science

Article Title: Aminopeptidase P3, a new member of the TNF-TNFR2 signaling complex, induces phosphorylation of JNK1 and JNK2.

doi: 10.1242/jcs.149385

Figure Lengend Snippet: Fig. 8. Induction of TNF-induced cell death by APP3 knockdown. (A) HEK293Tand HEK293T-TNFR2 cells cultured in a 96-well plate were treated with serially diluted TNF. After 24 hours, cell viability was measured by a WST-8 assay. Results are mean6s.d., n53. (B) HEK293T-TNFR2 cells were transfected with APP3 shRNA A, APP3 shRNA B or scrambled shRNA, and incubated for 48 hours. After cell lysis, suppression of APP3 was confirmed by western blotting. b-actin served as a loading control. (C) HEK293T-TNFR2 cells were transfected with APP3 shRNA A, APP3 shRNA B or scrambled shRNA. After 48 hours, these cells were treated with TNF (100 ng/ml) for 24 or 48 hours. Mock transfected cells were used as a reference. Cell viability was measured by a WST-8 assay. (D) HEK293T-TNFR2 cells pretreated with 100 mM apstatin were treated with TNF (100 ng/ml) for 24 or 48 hours. Cell viability was measured by WST-8 assay. Results are mean6s.d., n53.

Article Snippet: Two APP3 shRNA plasmids, each with a different target sequence (APP3 shRNA A, 59-AGACCAGACAGTGGTTGTGCTCTCCAACC39; and B, 59-GATGGAGGTTGTGAGTCTTCCTGCTATGT-39), and a scrambled negative control shRNA plasmid (OriGene) were transfected into the cells by lipofection.

Techniques: Knockdown, Cell Culture, Transfection, shRNA, Incubation, Lysis, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: An ANGPTL4–ceramide–protein kinase Cζ axis mediates chronic glucocorticoid exposure–induced hepatic steatosis and hypertriglyceridemia in mice

doi: 10.1074/jbc.RA118.006259

Figure Lengend Snippet: Myriocin (MYR) treatment reduced chronic dexamethasone exposure–induced hepatic steatosis and hypertriglyceridemia. WT and Angptl4−/− mice were treated with dexamethasone (0.84 mg/kg body weight) in drinking water for 7 days. Half of the mice received daily intraperitoneal injections of myriocin (0.5 mg/kg body weight) on days 4–7. A and B, at the end of treatment, the levels of TG in plasma (A) and liver (B) were measured. The error bars represent standard deviation (n = 6–8). *, p < 0.5. C, RNA from the liver of these mice was isolated, and the expression of genes involved in TG homeostasis was monitored by real-time PCR. The error bars represent S.E. (n = 7–9). *, p < 0.05. D, liver and epididymal white adipose tissue RNA was isolated, and gene expression for Angptl4 was measured. The error bars represent S.E. (n = 7–9). *, p < 0.05. WT mice were infected with adeno-associated virus (AAV8) expressing shRNA for scramble or Sptlc2 and were treated with dexamethasone for 2 weeks. E, liver was harvested and analyzed for decreased expression of Sptlc2. F and G, plasma (F) and liver (G) triglyceride levels were measured. The error bars represent standard deviation (n = 3–4). *, p < 0.05.

Article Snippet: AAV serotype 8 shRNA targeting Sptlc2 was purchased from Vector Biolabs.

Techniques: Clinical Proteomics, Standard Deviation, Isolation, Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Infection, Virus, shRNA

Journal: EMBO Molecular Medicine

Article Title: GDF 15 is a heart‐derived hormone that regulates body growth

doi: 10.15252/emmm.201707604

Figure Lengend Snippet: A Plasma IGF1 concentrations (ng/ml) in 7‐day‐old weight‐ and gender‐matched littermate WT mice injected with control or different proteins were measured by ELISA ( n = 3–5 mice per group, daily i.p. injection from 5 days of age). B–G Liver phosphorylated and total STAT5 and JAK2 as well as β‐actin (loading control) determined by Western blot (B); liver expression of Igf1 , Igfbp3, and Igfals quantified by qPCR (C); plasma IGF1 (D), IGFBP3 (E), and GH concentrations (F) measured by ELISA; and daily body weight (G) in weight‐ and gender‐matched littermate WT mice injected with control or GDF15 ( n = 5 per group, daily i.p. injection from 3 days of age). H Overnight‐fasted (in DMEM) WT mouse primary hepatocytes were first treated with different concentrations of GDF15 for 30 min and then with 20 ng/ml GH for 15 min. Cellular levels of phosphorylated STAT5, total STAT5, and β‐actin (loading control) were determined by Western blot. Data information: * P < 0.05, ** P < 0.01, and *** P < 0.001 between control and GDF15 by t ‐test. All values are mean + s.e.m. Source data are available online for this figure.

Article Snippet: AAV9‐Sico‐mouse Gdf15 shRNA (based on shAAV‐260008) or scramble control shRNA was custom‐built and manufactured by Vector Biolabs.

Techniques: Clinical Proteomics, Injection, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: EMBO Molecular Medicine

Article Title: GDF 15 is a heart‐derived hormone that regulates body growth

doi: 10.15252/emmm.201707604

Figure Lengend Snippet: Expression of Gdf15 in 3‐ ( n = 5–7 mice per group), 7‐ ( n = 6 mice per group), 10‐ ( n = 8–10 mice per group), and 13‐day‐old ( n = 12–13 mice per group) littermate mouse hearts quantified by qPCR. GDF15 protein level in 3‐, 7‐, 10‐, and 13‐day‐old littermate mouse hearts determined by Western blot. β‐Actin serves as a loading control. Plasma GDF15 concentrations in 3‐ ( n = 5–7 mice per group), 7‐ ( n = 6 mice per group), and 10‐day‐old littermate mice ( n = 8–10 mice per group) measured by ELISA. Representative pictures of 3‐, 7‐, 10‐, and 13‐day‐old littermate αHetγWT and αKOγKO mouse heart sections stained with GDF15 antibody (brown) and counterstained with hematoxylin (purple). Scale bar: 100 μm. Representative pictures of 16‐day‐old αKOγKO mouse hearts stained with GDF15 (red) and cardiac troponin I (green) antibodies. Arrows point to the nucleus of cardiomyocytes, and asterisks mark the nucleus of non‐cardiomyocytes. Scale bar: 20 μm. Data information: * P < 0.05, ** P < 0.01, and *** P < 0.001 between αKOγKO and all other littermate control genotypes by t ‐test. All values are mean + s.e.m.

Article Snippet: AAV9‐Sico‐mouse Gdf15 shRNA (based on shAAV‐260008) or scramble control shRNA was custom‐built and manufactured by Vector Biolabs.

Techniques: Expressing, Western Blot, Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Staining

Journal: EMBO Molecular Medicine

Article Title: GDF 15 is a heart‐derived hormone that regulates body growth

doi: 10.15252/emmm.201707604

Figure Lengend Snippet: A Design of AAV9‐mGDF15 shRNA construct to specifically knockdown GDF15 in Cre + cardiomyocytes. B–G Cardiac Gdf15 expression quantified by qPCR (B), plasma GDF15 concentrations measured by ELISA (C), cardiac Bnp expression quantified by qPCR (D), liver phosphorylated STAT5 level measured by ELISA (E), and plasma IGF1 (F) and GH concentrations (G) measured by ELISA in 9‐ to 10‐day‐old littermate control and αKOγKO mice ( n = 8–12 mice per group) that received pericardial injection of AAV9‐control or Gdf15 shRNA at 2 days of age. * P < 0.05, ** P < 0.01, and *** P < 0.001 by t ‐test. Values are mean + s.e.m.

Article Snippet: AAV9‐Sico‐mouse Gdf15 shRNA (based on shAAV‐260008) or scramble control shRNA was custom‐built and manufactured by Vector Biolabs.

Techniques: shRNA, Construct, Knockdown, Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Control, Injection

Journal: EMBO Molecular Medicine

Article Title: GDF 15 is a heart‐derived hormone that regulates body growth

doi: 10.15252/emmm.201707604

Figure Lengend Snippet: Plasma GDF15 concentrations in 2‐ to 3‐year‐old children diagnosed with heart disease with either normal body weight ( n = 35) or FTT ( n = 45) and in age‐ and gender‐matched healthy controls ( n = 45) were measured by ELISA. * P < 0.05 and *** P < 0.001 by t ‐test. Values are mean ± s.e.m. Cartoon illustrating how GDF15 and ANP/BNP relieve cardiac burden and coordinate cardiac function with the rest of the body.

Article Snippet: AAV9‐Sico‐mouse Gdf15 shRNA (based on shAAV‐260008) or scramble control shRNA was custom‐built and manufactured by Vector Biolabs.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay