Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 induces expression of proinflammatory cell adhesion molecules and monocyte chemoattractant protein-1 in human endothelial cells in a dose-dependent manner. (A) Representative Coomassie blue staining and Western blot analysis of purified recombinant human holo-RBP4 (1 μg per lane). Lane 1, Coomassie blue-stained gel; lane 2, Western blot with RBP4 antibody. (B) UV spectrum analysis of purified recombinant holo-RBP4 demonstrates a retinol/RBP4 ratio of approximately 0.9, indicating that the majority of the purified RBP4 consists of holo-RBP4. (C to F) Quantitative RT-PCR analysis for mRNA expression of VCAM-1, ICAM-1, E-selectin, and MCP-1 in human retinal capillary endothelial cells (HRCEC) treated with increasing concentrations of holo-RBP4 (10 to 100 μg/ml) or BSA or left untreated (Unt.). Significant differences compared to BSA treatment: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way analysis of variance (ANOVA) with Tukey's post hoc test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Expressing, Staining, Western Blot, Purification, Recombinant, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 activates NADPH oxidase protein expression and stimulates proinflammatory protein expression via an NADPH oxidase-dependent mechanism in HRCEC. (A and B) HRCEC were treated with BSA or increasing concentrations of holo-RBP4 for 24 h, and cell lysates were analyzed by Western blotting for Nox2 (A) and Nox4 (B). (C through H) HRCEC were pretreated with NADPH oxidase inhibitors DPI (20 μM) or apocynin (500 or 1,000 μM) for 2 h prior to the addition of holo-RBP4 at 100 μg/ml for 24 h. (C and D) Western blots of VCAM-1 following treatment with RBP4 in the absence or presence of DPI (C) or apocynin (D). (E through H) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (E), sICAM-1 (F), MCP-1 (G), and E-selectin (H) in HRCEC media. Significant differences compared to RBP4 treatment alone: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 increases cellular and extracellular protein levels of proinflammatory molecules in HRCEC in a dose- and time-dependent manner. (A to C) Western blots of VCAM-1 (A), E-selectin (B), and ICAM-1(C) from HRCEC treated with BSA or increasing concentrations of RBP4 for 24 h. (D to F) Western blots of VCAM-1 (D), E-selectin (E), and ICAM-1 (F) from HRCEC treated with RBP4 at 100 μg/ml for the indicated times of 0 to 48 h. (G to J) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (G), E-selectin (H), sICAM-1 (I), and MCP-1 (J) from HRCEC media following treatment with increasing concentrations of holo-RBP4 or BSA or no treatment (Unt.). (K to N) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (K), E-selectin (L), sICAM-1 (M), and MCP-1 (N) in HRCEC media following treatment with holo-RBP4 at 100 μg/ml for the indicated times of 0 to 48 h. Significant differences compared to BSA treatment: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 increases leukocyte adherence to human endothelial cells. Confluent monolayers of HRCEC were treated with either holo-RBP4 (100 μg/ml), BSA, or TNF-α (100 ng/ml) for 18 h. THP-1 monocytes were then added and cocultured for 3 h. (A) Representative phase-contrast images (magnification, ×20) of monocyte adherence to HRCEC after the indicated treatment. Adherent monocytes are apparent as small bright circular bodies above the HRCEC layer. (B) Adherent monocytes were counted per visual field at magnification of ×20. The graph shows the means ± standard deviations from 4 different visual fields for each treatment group (adherent leukocytes in TNF-α positive control were 205 ± 25 per visual field). ***, P < 0.001 versus BSA treatment by one-way ANOVA with Tukey's post hoc test. Untreat, untreated cells.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Positive Control

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 activates NF-κB and stimulates proinflammatory protein expression at least partially through an NF-κB-dependent mechanism in HRCEC. (A) Nuclear translocation of NF-κB p65 detected by immunocytochemistry in HRCEC after treatment with holo-RBP4 (100 μg/ml), BSA, or TNF-α for 12 h; untreat, untreated cells. (B) The relative nuclear transcriptional activity of NF-κB p65 was quantified using the ELISA-based TransAM NF-κB assay. HRCEC were treated with BSA or holo-RBP4 (100 μg/ml) for 6 h, and nuclear extracts were prepared and analyzed. Western blots of beta-actin, GAPDH, and fibrillarin demonstrate the enrichment and quality of nuclear extract preparations. Lane C, cytosolic extract; lane N, nuclear extract (10 μg protein/lane). (C) HRCEC were treated with either BSA or holo-RBP4 (100 μg/ml) for 24 h, and phosphorylation of NF-κB p65 at Ser536 was quantified by Western blotting and band densitometry analysis. (D through I) HRCEC were pretreated with NF-κB inhibitors PDTC (100 or 500 μM) or JSH-23 (10 or 50 μM) for 2 h prior to the addition of holo-RBP4 at 100 μg/ml for 24 h. (D through E) Western blots of VCAM-1 following treatment with RBP4 in the absence or presence of PDTC (D) or JSH-23 (E). (F through I) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (F), sICAM-1 (G), MCP-1 (H), and E-selectin (I) in HRCEC media. Significant differences compared to RBP4 treatment alone: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test. Significant difference compared to BSA treatment (panels B and C): †, P < 0.01 by Student's t test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Expressing, Translocation Assay, Immunocytochemistry, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4-mediated activation of NF-κB, NADPH oxidase, and proinflammatory molecules is retinol independent. (A) Quantitative RT-PCR analysis of STRA6 mRNA expression in HRCEC following 24 h of treatment with RBP4 (100 μg/ml) or BSA or no treatment (Untr.). STRA6 mRNA expression is shown relative to HPRT housekeeping gene expression and is 20-fold less abundant than HPRT. (B) STRA6 protein expression was undetectable by Western blotting of HRCEC. HEK-293A cells stably expressing STRA6 were also analyzed as a positive control. (C) HRCEC intracellular retinoid content following treatment with holo-RBP4 (100 μg/ml) or an equimolar amount (4.75 μM) of retinol, retinal, or retinoic acid. HRCEC did not uptake a significant amount of retinol from holo-RBP4. (D and E) Western blots of VCAM-1 and Nox2 (D) or Nox4 (E) in HRCEC treated with increasing concentrations of apo-RBP4 for 24 h. (F) HRCEC were treated with either BSA or apo-RBP4 (100 μg/ml) for 24 h, and phosphorylation of NF-κB p65 at Ser536 was quantified by Western blotting and band densitometry analysis. (G through J) ELISA-based quantification of soluble extracellular levels of MCP-1 (G), sVCAM-1 (H), sICAM-1 (I), and E-selectin (J) in HRCEC media following 24 h of treatment with increasing concentrations of either apo- or holo-RBP4 as indicated. Significant differences compared to BSA treatment: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Gene Expression, Western Blot, Stable Transfection, Positive Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 induces NF-κB, NADPH oxidase, and proinflammatory molecules in human umbilical vein endothelial cells. (A through C) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (A), sICAM-1 (B), and MCP-1 (C) in HUVEC media following 24 h of treatment with increasing concentrations of either apo- or holo-RBP4 as indicated. (D) Western blots of E-selectin and Nox2, VCAM-1, and Nox4 in HUVEC treated with increasing concentrations of holo-RBP4 for 24 h. (E) Nuclear translocation of NF-κB p65 detected by immunocytochemistry in HUVEC after treatment with holo-RBP4 (100 μg/ml), BSA, or TNF-α for 12 h. Untreat, untreated cells. (F) HUVEC were treated with either BSA or apo-RBP4 (100 μg/ml) for 24 h, and phosphorylation of NF-κB p65 at Ser536 was quantified by Western blotting and band densitometry analysis. Significant differences compared to BSA treatment: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test; †, P < 0.01 by Student's t test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Translocation Assay, Immunocytochemistry, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4-mediated induction of proinflammatory proteins in HUVEC is via activation of NADPH oxidase and NF-κB. (A through C) HUVEC were pretreated with NF-κB inhibitors PDTC (100 or 500 μM) or JSH-23 (10 or 50 μM) or with NADPH oxidase inhibitors DPI (20 μM) or apocynin (500 or 1,000 μM) for 2 h prior to the addition of holo-RBP4 at 100 μg/ml for 24 h. ELISA-based quantification of soluble extracellular levels of sICAM-1 (A), sVCAM-1 (B), and MCP-1 (C) in HUVEC media. Significant differences compared to RBP4 treatment alone: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA; †, P < 0.01 by Student's t test. (D and E) Western blots of VCAM-1 in HUVEC following RBP4 treatment in the presence or absence of PDTC (D) or DPI (E).

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Annals of clinical and translational neurology

Article Title: Proteomics and mathematical modeling of longitudinal CSF differentiates fast versus slow ALS progression.

doi: 10.1002/acn3.51890

Figure Lengend Snippet: Figure 6. Receiver operator characteristic (ROC) curves using targeted measurements of candidate markers of progression by ELISA in the (A) discovery and (B) validation cohorts. Respective area under the curve (AUC) for SERPINA4 (green), RBP4 (red), F12 (blue), and combination (purple) are provided.

Article Snippet: Measurements of human retinol binding protein 4 (RBP4) and kallistatin (SERPINA4) were performed using DuoSet ELISA kits (R&D systems; Minneapolis,per MN) following the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: Nature Communications

Article Title: Alteration of circadian machinery in monocytes underlies chronic kidney disease-associated cardiac inflammation and fibrosis

doi: 10.1038/s41467-021-23050-x

Figure Lengend Snippet: a Functional analysis of the genes using the KEGG database. Detailed data are presented in Supplementary Table . b , c The mRNA levels of Gpr68 in mouse primary cultured monocytes after treatment with TNFα, IL-6, angiotensin II, or retinol/RBP4 at the indicated concentrations for 24 h. d – f The protein levels of GPR68 and phosphorylated STAT5 (phos-STAT5), and the mRNA levels of Clock and Arntl in mouse primary cultured monocytes after treatment with 1 or 3 µM of retinol/RBP4 for 24 h. g Schematic representation of the upstream region of the mouse Gpr68 , Clock , and Arntl genes. The numbers indicate the distance from the transcription start site (+1). The circled letters (orange circles) indicate the location on the gene where each of the different primer sets localize for analysis of ChIP. h , i ChIP analysis of retinol/RBP4-induced changes in the binding amount of STAT5 protein to the upstream region of Clock and Arntl genes in mouse primary cultured monocytes or the collected monocytes prepared from Sham and 5/6Nx mice. The primer sets used qPCR are illustrated in g . j ChIP analysis of retinol/RBP4-induced changes in the binding amount of CLOCK and ARNTL protein to the upstream region of Gpr68 genes in mouse primary cultured monocytes. Using primer sets illustrated in Fig. . k Left panel shows the protein levels of STRA6 in monocytes transfected with siRNA against STRA6 (uncropped images are presented in Supplementary Fig. ). Right panels show STRA6-dependent phosphorylation of STAT5 and expression of GPR68, CLOCK, and ARNTL by serum from Sham and 5/6Nx mice in mouse primary cultured monocytes. For all panels, graphs show the mean ± SD of individual mice in independent experiments ( h , j n = 5; the others n = 4 for each group). Statistical significance was determined using one-way ANOVA with Dunnett’s post hoc tests ( c ), two-tailed Student’s t -tests ( d – f , h – j ), or two-way ANOVA with Tukey–Kramer post hoc tests ( k ). Numbers and P -values are shown in the graph. Source data are provided as a Source Data file.

Article Snippet: The calibration curve for calculating RBP4 concentrations was made by the quantified values of recombinant RBP4 protein levels (mouse; R&D systems, Inc., Minneapolis, MN, USA.

Techniques: Functional Assay, Cell Culture, Binding Assay, Transfection, Phospho-proteomics, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Alteration of circadian machinery in monocytes underlies chronic kidney disease-associated cardiac inflammation and fibrosis

doi: 10.1038/s41467-021-23050-x

Figure Lengend Snippet: a Correlation between serum retinol and RBP4 levels in 5/6Nx mice fed with normal or vitamin A-free diet. b Dietary deficiency of vitamin A suppresses the upregulation of Gpr68 , Clock , and Arntl mRNA levels in circulating monocytes. The mean value of the Sham group was set as 1.0. c Double immunofluorescence labeling of GPR68 with F4/80 in the ventricle slices prepared from 5/6Nx mice fed with normal or Vitamin A-free diet. High-GPR68-expressing cells are double-labeled (yellow) with F4/80. The scale bar indicates 50 μm. d Serum BNP concentrations in Sham and 5/6Nx mice fed with normal or vitamin A-free diet. e Dietary deficiency of vitamin A ameliorates CKD-induced cardiac fibrosis. The Masson’s trichrome staining show tissue fibrosis in blue. Scale bars indicate 1 mm (upper panel) and 50 μm (lower panel). For all panels, graphs show the mean ± SD of individual mice in independent experiments. Statistical significance was determined using one-way ANOVA with Tukey–Kramer post hoc tests ( a , b , d ). Numbers and P -values are shown in each graph. Source data are provided as a Source Data file.

Article Snippet: The calibration curve for calculating RBP4 concentrations was made by the quantified values of recombinant RBP4 protein levels (mouse; R&D systems, Inc., Minneapolis, MN, USA.

Techniques: Immunofluorescence, Labeling, Expressing, Staining

Journal: Nature Communications

Article Title: Alteration of circadian machinery in monocytes underlies chronic kidney disease-associated cardiac inflammation and fibrosis

doi: 10.1038/s41467-021-23050-x

Figure Lengend Snippet: a Dose-dependent increase in the expression of GPR68 mRNA (left) and its protein (right) in human primary cultured monocytes by treatment with retinol/RBP4 for 24 h. b The phosphorylation state of STAT5 in human primary cultured monocytes after treatment with retinol/RBP4. c Schematic representation of the upstream region of the human GPR68 , CLOCK , and ARNTL genes. The circled letters (green circles) indicate the location on the gene where each of the different primer sets localize. d The binding of CLOCK, ARNTL, and STAT5 to regions, described in c , in human (healthy) primary cultured monocytes after treatment with retinol/RBP4 for 24 h. The primer sets used in qPCR are illustrated in c . e LPS-stimulated release of TNFα and IL-6 from human primary cultured monocytes after treatment with retinol/RBP4 for 24 h. f Serum retinol and RBP4 levels in CKD patients. The upper panel shows relationship between retinol and RBP4 levels. Lower panels show these levels stratified by serum creatinine levels (Scr). g , h Relationship retinol levels to mRNA expressions of GPR68 , IL-6 , TNFα , CLOCK , or ARNTL in human primary cultured monocytes incubated in media containing 20% serum collected from healthy subjects and CKD patients stratified by Scr. The release of TNFα and IL-6 from human primary cultured monocytes were assessed after incubation in the media containing 20% serum for 24 h. i Comparison of the values stratified by BNP concentrations. Left panel shows the expression levels of GPR68 mRNA in human primary cultured monocytes incubated same as in g . Right panel shows serum retinol levels. For panel f – i , each plot shows a value obtained using an individual human serum. For all panels, graphs show the mean ± SD in independent experiments ( a , b , d , e n = 5 for each group, f – i shown in each graph). Statistical significance was determined using one-way ANOVA with Dunnett’s post hoc tests ( a ), two-tailed Student’s t -tests ( a , b , d , g ), or one-way ANOVA with Tukey–Kramer post hoc tests ( e , i ). Numbers and P -values are shown in each graph.

Article Snippet: The calibration curve for calculating RBP4 concentrations was made by the quantified values of recombinant RBP4 protein levels (mouse; R&D systems, Inc., Minneapolis, MN, USA.

Techniques: Expressing, Cell Culture, Phospho-proteomics, Binding Assay, Incubation, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Alteration of circadian machinery in monocytes underlies chronic kidney disease-associated cardiac inflammation and fibrosis

doi: 10.1038/s41467-021-23050-x

Figure Lengend Snippet: Serum levels of retinol and RBP4 were increased during chronic renal failure. The retinol-bound RBP4 activates JAK2/STAT5 signaling in circulating monocytes through STRA6 and induces the expression of GPR68 via activation of CLOCK/ARNTL. The high-GPR68-expressing monocytes infiltrate into the heart potential for producing inflammatory cytokines and their cardiac infiltration exacerbates inflammation and fibrosis.

Article Snippet: The calibration curve for calculating RBP4 concentrations was made by the quantified values of recombinant RBP4 protein levels (mouse; R&D systems, Inc., Minneapolis, MN, USA.

Techniques: Expressing, Activation Assay

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Retinol-Binding Protein 4 Induces Hepatic Mitochondrial Dysfunction and Promotes Hepatic Steatosis.

doi: 10.1210/jc.2016-1320

Figure Lengend Snippet: Figure 1. Aberrant elevation of RBP4 expression in NAFLD patients and mice (A-D left) Liver samples from NAFLD patients and healthy controls were collected and subjected to analysis. (A-D right) ApoE-/- mice were kept on a high fat and high cholesterol diet to induce the full spectrum of liver pathology that characterizes human NAFLD before liver samples were collected and subjected to analysis. A, RBP4 mRNA expression in liver tissues of NAFLD patients (left) and mouse models (right). Results were expressed as fold of control. B, Correlation between hepatic triglyceride contents and RBP4 mRNA levels in NAFLD patients (left) and mouse models (right). C, Immunohistochemistry with anti-RBP4 was performed on human liver tissues (left) and mouse models (right). RBP4 positive cells were visualized by DAB staining and Hematoxylin was used as counterstaining. Magnification 400. D, Representative Western blot analysis of RBP4 protein expression from NAFLD patients (left) and mouse models (right). Each lane represented the pool of 3 subjects or mice and the NAFLD patients were all from the grade 2–3 group. **P .01, ***P .001 between Control and NAFLD patients. ##P .01 between Control and NAFLD mice.

Article Snippet: Transgenic mice expressing human RBP4 on a C57BL/6J background were constructed by Cyagen Biosciences as described (24) with modification.

Techniques: Expressing, Control, Immunohistochemistry, Staining, Western Blot

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Retinol-Binding Protein 4 Induces Hepatic Mitochondrial Dysfunction and Promotes Hepatic Steatosis.

doi: 10.1210/jc.2016-1320

Figure Lengend Snippet: Figure 2. RBP4 overexpression accelerates the progression of hepatic steatosis in mice. Both RBP4-Tg and the WT littermates were fed RC or HFD for 16 wks and fasted overnight prior to analysis. A, Representative histological sections stained with hematoxylin-eosin (Magnification 400, black arrow indicates ballooned cells). B, Oil Red O staining of liver sections (Magnification 400). C, Triglyceride levels in the liver. D, Masson trichrome staining of liver sections (Magnification 200, black arrow indicates collagen). E, Quantitative RT-PCR analysis of genes involved in fibrogenesis (Col1a1, TGF-, Timp1) in the livers. F, Quantitative RT-PCR analysis of genes involved in inflammation (TNF-, MCP- 1, IL-1, IL-6) in livers. G, Histological scoring of NAFLD in liver sections. H, Serum levels of ALT and AST. Data were expressed as the mean SEM (n 8–10 each group). ***P .001 RBP4-Tg/HFD vs WT/HFD; #P .05, ##P .01, ###P .001 RBP4-Tg/RC vs WT/RC.

Article Snippet: Transgenic mice expressing human RBP4 on a C57BL/6J background were constructed by Cyagen Biosciences as described (24) with modification.

Techniques: Over Expression, Staining, Quantitative RT-PCR

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Retinol-Binding Protein 4 Induces Hepatic Mitochondrial Dysfunction and Promotes Hepatic Steatosis.

doi: 10.1210/jc.2016-1320

Figure Lengend Snippet: Figure 3. RBP4 overexpression induces mitochondrial dysfunction in mice. RBP4-Tg and the WT littermates were fed RC or HFD for 16 wks before analysis. A, Liver gDNA was isolated and mtDNA content was quantified. B, Representative Western blot analysis of CPS-1 in isolated mitochondria. C, Representative electron micrographs from livers of RC and HFD-fed mice (Magnification 15 000) Swollen mitochondria with disrupted membranes (black arrows) and pale matrix (white asterisks). D, Citrate synthase activity, (E) Quantitative RT-PCR analysis of UCP-2 levels in the liver, (F) ATP quantification, (G) CPT-1 activity, (H) Quantitative RT-PCR analysis of genes involved in mitochondrial oxidation (CPT1, PPAR) and (I) hepatic -HAD activity of liver mitochondria isolated from mice after 16-week feeding. Data were expressed as the mean SEM (n 8–10 each group). *P .05, **P .01, ***P .001 RBP4-Tg/HFD vs WT/HFD; #P .05, ##P .01, ###P .001 RBP4-Tg/RC vs WT/RC.

Article Snippet: Transgenic mice expressing human RBP4 on a C57BL/6J background were constructed by Cyagen Biosciences as described (24) with modification.

Techniques: Over Expression, Isolation, Western Blot, Activity Assay, Quantitative RT-PCR

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Retinol-Binding Protein 4 Induces Hepatic Mitochondrial Dysfunction and Promotes Hepatic Steatosis.

doi: 10.1210/jc.2016-1320

Figure Lengend Snippet: Figure 4. RBP4 overexpression induces hepatic LCAD hyperacetylation and suppresses SIRT3 activity in mice. RBP4-Tg and the WT littermates were fed RC or HFD for 16 wks before analysis. A, Representative Western blot analysis of acetylation level of LCAD. B, Hepatic LCAD activity. C, Quantitative RT-PCR analysis of hepatic SIRT3 expression. D, Representative Western blot analysis of SIRT3 in isolated liver mitochondria. E, Hepatic SIRT3 activity. Data were expressed as the mean SEM (n 10 each group). **P .01, ***P .001 RBP4-Tg/HFD vs WT/HFD; ##P .01, ###P .001 RBP4- Tg/RC vs WT/RC.

Article Snippet: Transgenic mice expressing human RBP4 on a C57BL/6J background were constructed by Cyagen Biosciences as described (24) with modification.

Techniques: Over Expression, Activity Assay, Western Blot, Quantitative RT-PCR, Expressing, Isolation

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Retinol-Binding Protein 4 Induces Hepatic Mitochondrial Dysfunction and Promotes Hepatic Steatosis.

doi: 10.1210/jc.2016-1320

Figure Lengend Snippet: Figure 5. SIRT3 inhibition mediates LCAD hyperacetylation induced by RBP4. HepG2 cells were transfected with control or SIRT3 plasmid for 36h followed by RBP4 treatment (60 g/mL) for another 24 hours before analysis. A, Efficiency of SIRT3 transfection was determined by Western blot. B, Representative western blot analysis of acetylation level of LCAD and (C) LCAD activity in cells. D, Coimmunoprecipitation of SIRT3-LCAD complex. Coimmunoprecipitation was carried out with lysates treated with or without RBP4 (60 g/ml) stimulation and following lysis, SIRT3-containing proteins immunoprecipitated with a polyclonal anti-SIRT3 antibody and then immunoblotted using an anti-LCAD antibody. E, Citrate synthase activity, (F) ATP levels, (G) CPT-1 activity and (H) -HAD activity in HepG2 cells were determined. *P .05, **P .01, ***P .001 between RBP4 stimulated Control and SIRT3 plasmid transfected cells. n 4–5 independent experiments for each assay.

Article Snippet: Transgenic mice expressing human RBP4 on a C57BL/6J background were constructed by Cyagen Biosciences as described (24) with modification.

Techniques: Inhibition, Transfection, Control, Plasmid Preparation, Western Blot, Activity Assay, Lysis, Immunoprecipitation

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: Dynamic expression of Rbp4 in mouse ovaries during the different stages of development and the estrous cycle. (A) Real-time qPCR analyses of Rbp4 mRNA levels in mouse ovaries during different developmental stages. Data are presented as means ± SEM, n = 8. (B) Relative Rbp4 mRNA levels in mouse ovaries during diestrus (D), proestrus (P), estrus (E), and metestrus (M). Data are presented as mean ± SEM, n = 8. RBP4 protein levels in the ovaries of mice during the estrous cycle were determined via Western blot analyses (D), and blots were quantified via densitometry (C). The protein levels were normalized to GAPDH. Data are presented as means ± SEM, n = 3. Means with different letters represent a significant difference at the 0.05 level

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Expressing, Western Blot

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: Effects of gonadotropins and ovarian steroids on the expression of Rbp4 in the 3-week-old mouse ovary. (A) Rbp4 mRNA expression levels in the ovaries of mice treated with FSH, LH, or FSH + LH for 48 h. Data are presented as means ± SEM, n = 8. (B) Rbp4 mRNA expression levels in the ovaries of mice treated with 17β-estradiol for 24 or 48 h. Data are presented as mean ± SEM, n = 8. RBP4 protein levels in the ovaries of mice treated with FSH or LH were determined using Western blot analyses (D), and blots were quantified via densitometry (C). The protein levels were normalized to GAPDH. Data are presented as means ± SEM, n = 3. Means with different letters represent a significant difference at the 0.05 level

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Expressing, Western Blot

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: Localization of RBP4 protein in the ovaries of the immature mice (3 weeks of age) treated with or without FSH for 48 h. Negative controls (NC), which were incubated with 5% goat serum (NC1) or PBS (NC2) instead of the primary antibody, were used to validate the specificity of RBP4 antibody. Positive signals for RBP4 were detected in granulosa and theca cell layers. Bar = 40 μm

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Incubation

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: Effects of gonadotropins on the expression of Rbp4 in mouse granulosa cells. (A) Relative Rbp4 mRNA levels in mouse granulosa cells treated with FSH and LH for 24 h. Data are presented as means ± SEM, n = 4. RBP4 protein levels in mouse granulosa cells treated with FSH and LH for 24 h were determined using Western blot analyses (C), and blots were quantified via densitometry (B). The protein levels were normalized to GAPDH. Data are presented as means ± SEM, n = 3. Means with different letters represent a significant difference at the 0.05 level

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Expressing, Western Blot

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: PKA mediates the FSH induction of Rbp4 in mouse granulosa cells. (A) Activation of PKA by cAMP and FSH increased Rbp4 mRNA levels. Data are presented as means ± SEM, n = 4. (B) The PKA inhibitor H-89 abrogated FSH induced Rbp4 mRNA expression. Granulosa cells were pre-incubated with 10 μM H-89 for 1 h, followed by incubation with 100 IU/L FSH for 24 h. Data are presented as means ± SEM, n = 4. (C) Densitometric quantification of Western blots for RBP4 (D). The protein levels were normalized to GAPDH. Data are presented as means ± SEM, n = 3. Means with different letters represent a significant difference at the 0.05 level

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Activation Assay, Expressing, Incubation, Western Blot

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: HMGA1, SF-1, and LRH-1 are involved in the FSH induction of Rbp4 in mouse granulosa cells. mRNA (A) and protein (D) levels of Hmga1 in mouse granulosa cells transfected with 50 nM Hmga1 siRNA. The mRNA (B) and protein (E) levels of Sf-1 in mouse granulosa cells transfected with 50 nM Sf-1 siRNA. The mRNA (C) and protein (F) levels of Lrh-1 in mouse granulosa cells transfected with 50 nM Lrh-1 siRNA. The mRNA levels were determined using real-time PCR. Data are presented as means ± SEM, n = 4. The protein levels were determined using Western blot analyses, and blots were quantified via densitometry. The protein levels were normalized to GAPDH. Data are presented as means ± SEM, n = 3. *p < 0.05, ** p < 0.01. (G) The mRNA levels of Rbp4 in mouse granulosa cells transfected with NC siRNA, Hmga1 siRNA, Sf-1 siRNA, or Lrh-1 siRNA (50 nM). Data are presented as means ± SEM, n = 4. (H), (I), and (J) The RBP4 protein levels in mouse granulosa cells transfected with NC siRNA, Hmga1 siRNA, Sf-1 siRNA, or Lrh-1 siRNA (50 nM). The protein levels were determined using Western blot analyses and blots were quantified via densitometry. The protein levels were normalized to GAPDH. Data are presented as means ± SEM, n = 3. Means with different letters represent a significant difference at the 0.05 level

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Western Blot

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: Schematic overview of the proposed pathway involved in the induction of Rbp4 by FSH. FSH binds to its receptor (FHR) and elevates cAMP levels, which activates PKA. PKA then stimulates the expression of HMGA1, SF-1, and LRH-1, and promotes Rbp4 transcription

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Expressing