Journal: American journal of physiology. Endocrinology and metabolism

Article Title: The transcriptional coregulator CITED2 suppresses expression of IRS-2 and impairs insulin signaling in endothelial cells.

doi: 10.1152/ajpendo.00435.2020

Figure Lengend Snippet: Figure 1. CITED2 selectively inhibits insulin signaling in endothelial cells in vitro and in vivo. A–E: lung endothelial cells were isolated from Cited2fl/flmice and treated with adenovirus expressing cre recombinase (Ad-Cre) or GFP only (Ad-GFP), thereby creating CITED2 knockout (CITED2 KO) and control cultures from the same cell isolation. A: CITED2 expression was measured in cell lysate by Western blotting. B: cell cultures were serum-starved over- night and treated with 10 nM insulin for 5 min. Protein expression was measured in cell lysate by Western blotting. C–E: quantitative analysis of insulin- stimulated phosphorylation of insulin receptor-b (INSRb), Akt, and ERK based on densitometry of 3–4 independent experiments and normalization to the baseline phosphorylation in control cells. F–G: Cdh5-cre Cited2fl/fland Cited2fl/flcontrol mice were injected intravenously with 5 U insulin or vehicle. F: after 5 min, the aorta was isolated and flash-frozen. Levels of Akt, phosphorylated Akt, and b-actin were measured by Western blotting of tissue lysate. G: quantitative analysis of insulin-stimulated Akt phosphorylation based on densitometry of 5 pairs of animals. P < 0.05, P < 0.01, P < 0.001, 2- way ANOVA followed by Tukey’s multiple-comparisons test. CITED2, CBP/p300 interacting transactivator-2; ERK, extracellular signal-related kinase; GFP, green fluorescent protein.

Article Snippet: Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed as previously described (15) with an anti-p300 antibody (NB100616, Novus Biologicals).

Techniques: In Vitro, In Vivo, Isolation, Expressing, Knock-Out, Control, Cell Isolation, Western Blot, Phospho-proteomics, Injection

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: The transcriptional coregulator CITED2 suppresses expression of IRS-2 and impairs insulin signaling in endothelial cells.

doi: 10.1152/ajpendo.00435.2020

Figure Lengend Snippet: Figure 4. CITED2 overexpression recapitulates endothelial insulin resistance. A–C: MS1 endothelial cells were cultured with lentivirus expressing FLAG- CITED2 for 12 h, and then, lentiviral medium was replaced with complete medium for 48h. Stably transduced MS1 cells were selected with puromycin (0.1%) for 72h. A: control and FLAG-CITED2-overexpressing cell cultures were serum-starved overnight and treated with 10 nM insulin for 5 min. Protein levels were measured in cell lysate by Western blotting. B: quantitative analysis of insulin-stimulated phosphorylation of Akt based on densitometry of 4 independent experiments. C: control and FLAG-CITED2-overexpressing cultures were serum-starved overnight and treated with 10 nM insulin for 4 h. Vegfa expression was measured by real-time PCR of 3 independent experiments, and relative Vegfa mRNA expression was determined by normaliza- tion to the housekeeping gene Rplp0. P < 0.05, P < 0.01, 2-way ANOVA followed by Tukey’s multiple-comparisons test. CITED2, CBP/p300 interact- ing transactivator-2.

Article Snippet: Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed as previously described (15) with an anti-p300 antibody (NB100616, Novus Biologicals).

Techniques: Over Expression, Cell Culture, Expressing, Stable Transfection, Control, Western Blot, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: The transcriptional coregulator CITED2 suppresses expression of IRS-2 and impairs insulin signaling in endothelial cells.

doi: 10.1152/ajpendo.00435.2020

Figure Lengend Snippet: Figure 5. CITED2 limits HIF-2a-mediated transcription of Irs2. A: MS1 endothelial cells transduced with lentivirus expressing FLAG-CITED2 were cotrans- fected with Renilla luciferase and normoxia-stable pcDNA3.1 mHIF-2a MYC P405A/P530V/N851A (HIF2a-DNA3.1), Irs2 promoter reporter (Irs2-pGL3), or empty vector control (pGL3, pcDNA3.1) plasmids. After 24h, cells were lysed and luminescent signal measured with the Dual Luciferase Reporter Gene Assay kit. B: CITED2 KO and control endothelial cells were cross-linked, lysed, and sonicated to produce 200–1,000-bp chromatin fragments as ana- lyzed by agarose gel electrophoresis. Immunoprecipitations were performed with 10 μg of p300 antibody (NB100-616, Novus Biologicals) or mouse IgG. Relative enrichment of chromatin was determined by real-time PCR. C: CITED2 inhibition of the p300/CBP-HIF-2a transcriptional complex decreases Irs2 transcription and IRS-2 expression, leading to impaired insulin signaling in endothelial cells. P < 0.01, Student’s t test. P < 0.001, P < 0.0001, 1-way ANOVA followed by Tukey’s multiple-comparisons test. CBP, CREB-binding protein; CITED2, CBP/p300 interacting transactivator-2; ChIP, chromatin immunoprecipitation; HIF, hypoxia-inducible factor; HRE, hypoxia-response element; INSR, insulin receptor.

Article Snippet: Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed as previously described (15) with an anti-p300 antibody (NB100616, Novus Biologicals).

Techniques: Transduction, Expressing, Luciferase, Plasmid Preparation, Control, Reporter Gene Assay, Sonication, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Inhibition, Binding Assay, Chromatin Immunoprecipitation