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: 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: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have 10- to 6-fold increases in serum RBP4 levels and maintain a normal body mass and fasting blood glucose, serum insulin, and triglyceride levels. (A) Total (mouse and human transgene) RBP4 levels in mouse serum (n ≥ 4 mice per age-genotype combination; WT, wild type). (B) TTR levels in mouse serum at 2 months of age (n ≥ 5 mice per genotype; data were analyzed by Student's t test). (C and D) Blood glucose levels in mice that had fasted for 6 h (n ≥ 6 mice per measurement). (E and F) Body mass measurements in mice that had fasted for 6 h (n ≥ 6 mice per measurement). (G) Insulin levels in the sera of 9-month-old mice that had fasted for 6 h (no significant difference between genotypes was detected on the basis of Student's t test). (H) Triglyceride levels in serum from 4-month-old mice that had fasted for 6 h (no significant difference between genotypes was detected on the basis of Student's t test). (I) Adiponectin levels in serum from 4-month-old mice that had fasted for 6 h (no significant difference between genotypes was detected on the basis of Student's t test). (J and K) Results of insulin tolerance tests in which male mice aged 17 to 21 weeks (n = 7 to 8 mice per genotype) were fasted for 6 h and then given a single i.p. injection of insulin (0.75 unit/kg) at time zero, followed by blood glucose measurements at 0, 15, 30, 60, 90, and 120 min postinjection. All graph values are means ± SEMs. **, P < 0.01 by Student's t test; ***, P < 0.001 by Student's t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Injection

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: Mouse serum levels of retinol and RBP4 a

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have age-dependent inner retinal degeneration. (A and B) Quantitative histological analyses of the total retina, INL, and ONL thickness for retinas from mice aged 1 month (A) or 6 months (B) (WT, wild-type; ONH, optic nerve head). Values are means ± SEMs for ≥8 mice for each data point. *, P < 0.05 by one-way analysis of variance (ANOVA) with Tukey's post hoc test; **, P < 0.01 by one-way ANOVA with Tukey's post hoc test; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test. (C to E) Representative confocal images of retinal paraffin sections from 9-month-old mice immunofluorescently labeled for rhodopsin (C), cone S-opsin (D), cone M-opsin (E). (F) Representative confocal images of retinal cryosections from 6-month-old mice immunofluorescently labeled for cone arrestin. DAPI counterstaining is shown in blue. Confocal images were acquired under a 63× magnification objective, but rhodopsin images are shown with an additional digital zoom of ×2. All images shown represent the maximum stack of z-plane acquisition. All images were acquired from central retina sections and are representative of those from 4 to 6 independent samples from mice of each genotype. Bars, 10 μm (C to E) and 20 µm (F).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Labeling

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: Visual acuity is significantly reduced in RBP4-Tg mice. (A to C) RBP4 and albumin levels in perfused retinas from mice (WT, wild type) aged 6 months (n ≥ 5 mice per genotype); (D and E) OKT response to changes in spatial frequency; (F and G) OKT response to changes in grating contrast. All OKT analyses were performed on ≥6 mice per genotype. Values are means ± SEMs. n.s., not significant by Student's t test; *, P < 0.05 by Student's t test; **, P < 0.01 by Student's t test; ***, P < 0.001 by Student's t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have an age-related loss of rod and cone bipolar cells. (A to G) Representative confocal images of retinal cryosections from 6-month-old mice immunofluorescently labeled for distinct INL cell types, as follows: all bipolar cells (green, Chx10) and rod bipolar cells (red, PKC-α) (A), rod bipolar cells (PKC-α) (B), cone bipolar cells (secretagogin) (C), calcium-binding amacrine and ganglion cells (calretinin) (D), glycinergic amacrine cells (GLYT1) (E), horizontal cells (calbindin) (F), and Müller cells (glutamine synthetase [GS]) (G). (H) Representative confocal images of retinal whole mounts immunolabeled with NeuN (green) to stain retinal ganglion cell bodies and beta-III tubulin (red) to stain ganglion axonal processes. In each panel, the image on the left is from a wild-type mouse retina and the image on the right is from an RBP4-Tg mouse retina. Confocal images were acquired under a 63× (A to G) or 40× (H) magnification objective, but the secretagogin (C) and calbindin (F) images are shown with an additional digital zoom of ×2. All images shown represent the maximum stack of z-plane acquisition. All images were acquired from central retina sections and are representative of those from 3 to 7 independent samples from mice of each genotype. Bars, 10 μm.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Labeling, Binding Assay, Immunolabeling, Staining

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have early-onset retinal gliosis and microglia activation. (A and B) Representative confocal images of retinal cryosections from 1-month-old mice (A) and 6-month-old (B) mice immunofluorescently labeled for GFAP, a marker of gliosis (WT, wild type). DAPI counterstaining is shown in blue. Confocal images were acquired under a 40× magnification objective and represent the maximum stack of z-plane acquisition. All images were acquired from central retina sections and are representative of those from 3 to 5 independent samples from mice of each genotype. Bars, 10 μm. (C and D) Representative confocal images of retinal whole mounts from 1-month-old mice immunofluorescently labeled for microglia marker CD11b. Confocal images were acquired under a 40× magnification objective, and each image represents a single layer of z-plane acquisition. Arrows, microglia cells with enlarged cell bodies characteristic of activated microglia. All images were acquired from the midperiphery and are representative of those from 3 to 6 independent samples from mice of each genotype. Bars, 40 μm. (E and F) Microglia cell bodies were counted in retinal whole mounts under a 40× magnification objective. Each field of view corresponds to a surface area of 0.14 mm2. Microglia were counted in 3 to 5 retinas per genotype. Values are means ± SEMs. **, P < 0.01 by Student's t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Activation Assay, Labeling, Marker

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have dominant and progressive inner retinal dysfunction leading to secondary photoreceptor dysfunction. (A and B) Scotopic ERG a-wave and b-wave amplitudes in mice aged 1 to 9 months (WT, wild type). (C) Photopic ERG b-wave amplitudes in mice aged 1 to 9 months. Values are means ± SEMs for ≥6 mice per age-genotype combination. n.s., not significant by one-way analysis of variance (ANOVA) with Tukey's post hoc test; *, P < 0.05 by one-way ANOVA with Tukey's post hoc test; **, P < 0.01 by one-way ANOVA with Tukey's post hoc test; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test. (D) Representative scotopic ERG responses to increasing light intensities (0.004 to 40 cd · s/m2) in mice aged 6 months. Note the pronounced b-wave decline compared to the more subtle a-wave reduction.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have deficiencies in photoreceptor ribbon synapses and cone bipolar terminals. (A to E) Representative confocal images of retinal cryosections from 1-month-old (A) or 6-month-old (B to E) mice (WT, wild type) immunolabeled for presynaptic photoreceptor ribbon synapses (red, bassoon; green, ribeye) (A and B), photoreceptor ribbon synapse connection to postsynaptic rod bipolar cell dendrites (red, bassoon; green, PKC-α) (C), photoreceptor ribbon synapse connection to postsynaptic horizontal cell dendrites (red, bassoon; green, calbindin) (D), and photoreceptor presynaptic terminals (red, VLGUT1) and cone bipolar axonal and dendritic terminals (green, ZNP-1) (E). Confocal images were acquired under a 63× magnification objective, but panels A to C are shown with an additional digital zoom of ×4, and panel D is shown with an additional zoom of ×6. All images shown represent the maximum stack of z-plane acquisition. DAPI counterstaining is shown in blue. Arrowheads, displaced VGLUT1; arrows, thinning or loss of ZNP-1 labeling. All images were acquired from central retina sections and are representative of those from 4 to 8 independent samples from mice of each genotype. Bars, 10 μm.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Immunolabeling, Labeling

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have normal levels of retinal visual cycle retinoids and bisretinoids A2E and iso-A2E. (A to C) HPLC-based quantification of visual cycle retinoid isomers in eyes from 2- to 11-month-old mice dark adapted for 16 h prior to euthanization. Values are means ± SEMs for 4 mice per genotype-age combination. ns, not significant. P values were determined by Student's t test. (D) HPLC-based quantification of bisretinoid A2E and iso-A2E levels in eyecups from 9-month-old mice reared in cyclic room light. Values are means ± SEMs for 4 samples (4 eyecups per sample from 8 mice total) per genotype; n.s., not significant by Student's t test. (E) Quantitative RT-PCR was performed to measure RARβ mRNA levels in retinas from mice aged 3 months. Values are means ± SEMs for 3 samples per group. ns, not significant by Student's t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have normal retinal stratification and RPE pigmentation and integrity. (A) Representative retinal fundus images of wild-type (WT) and RBP4-Tg mice aged 6 months. (B) Hematoxylin- and eosin-stained retinal sections from mice aged 6 months. Magnifications, ×40; bars, 20 μm. (C) Bright-field images of retinal whole mounts showing normal RPE pigmentation in 6-month-old mice. Magnifications, ×4. (D) Representative confocal images of RPE whole mounts from 6-month-old mice immunofluorescently labeled for ZO-1 to demarcate tight junctions. Confocal images were acquired under a 63× magnification objective and represent the maximum stack of z-plane acquisition. Bars, 20 μm. The images in panels B and D were acquired from central retina sections. All images are representative of those from 4 to 6 independent samples from mice of each genotype.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Staining, Labeling

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have normal retinal vasculature. (A) Fluorescein angiogram images from wild-type (WT) and RBP4-Tg mice aged 6 months. Each image shown is from a different mouse. (B) Epifluorescent images from whole-mount retinas following a retinal leukostasis procedure in which mice were perfused with FITC-concanavalin A to label adherent leukocytes. Arrows, adherent leukocytes. Magnification, ×20. (C) Quantification of retinal leukostasis. Values are means ± SEMs for ≥3 mice (6 eyes) per genotype-age combination. There was no statistically significant difference between genotypes by two-way analysis of variance.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: Microglia projections extend into the outer nuclear layer in RBP4-Tg mice. (A and B) Representative confocal images of retinal whole mounts from 6-month-old mice immunofluorescently labeled for the microglia marker Iba-1. Confocal images were acquired under a 63× magnification objective, and each image represents the z-series of the inner plexus layer (GCL, ganglion cell layer). Numbered boxes denote cells that are shown at higher magnification to the right side of the original image. All images were acquired from the midperiphery and are representative of those from 3 to 5 independent samples from mice of each genotype. Note the enlarged cell bodies in the microglia of RBP4-Tg mouse retinas. Bars, 40 μm. (C and D) Representative 3D confocal images of retinal whole mounts from 6-month-old mice immunofluorescently labeled for CD31 (red) and either Iba-1 (green) (C) or CD11b (green) (D). All images were acquired from the midperiphery and are representative of those from 3 to 5 independent samples from mice of each genotype. Note the microglia extensions into the ONL in the retinas of RBP4-Tg mice only.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Labeling, Marker

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have retinal neuroinflammation. qRT-PCR analyses showed increased levels of pro-IL-18 (A) and reduced levels of pro-IL-1β (B) mRNA expression in retinas from 6-month-old RBP4-Tg mice. (C) Western blots show no retinal expression of mature IL-1β in RBP4-Tg or wild-type mice at age 3 months. Lane +C, 1 μg recombinant mouse IL-1β as a positive control. The upper band near 45 kDa is β-actin, while the lower band near 15 kDa is mature IL-1β. (D and E) Western blot and densitometry (densitomet) analyses show significantly increased expression of mature IL-18 in retinas from RBP4-Tg mice at age 6 months. Densitometry is from 3 independent experiments consisting of 3 mice per genotype. (F) Serum IL-18 levels measured by ELISA. (G) The enzymatic activity of caspase-1 in lysates of retinas from 6-month-old mice was determined by incubation with the colorimetric substrate YVAD-pNA. (H) qRT-PCR analyses show decreased levels of NLRP3 mRNA expression in retinas from 6-month-old RBP4-Tg mice. Values are means ± SEMs for 4 to 6 mice per genotype. n.s., not significant by Student's t test; *, P < 0.05 by Student's t test; **, P < 0.01 by Student's t test; ***, P < 0.001 by Student's t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Recombinant, Positive Control, Enzyme-linked Immunosorbent Assay, Activity Assay, Incubation

Journal: Scientific reports

Article Title: Retinol binding protein 4 abundance in plasma and tissues is related to body fat deposition in cattle.

doi: 10.1038/s41598-019-44509-4

Figure Lengend Snippet: Figure 1. Fold changes of RBP4 mRNA abundance in different adipose tissues, liver and muscle of high carcass fat (HCF, n = 20) compared to low carcass fat (LCF, n = 18) bulls. Data were normalized to beta-2- microglobulin (B2M) and ubiquitously expressed transcript (UXT) for adipose tissues, UXT and ribosomal protein S9 (RPS9) for liver tissue, and topoisomerase II beta (TOP2B) and B2M for muscle tissue and calculated using the REST software (Version 2.0.13, QIAGEN, Hilden, Germany). Graphs show fold changes of expression of HCF vs. LCF bulls with standard errors calculated by REST software45. * indicate significant differences between groups with P < 0.05. SCF – subcutaneous fat, PF – perirenal fat, OF – omental fat, IF – intestinal fat, MLD – Musculus longissimus dorsi.

Article Snippet: After blocking with 10% goat serum in PBST for 15 min, sections were incubated with the Biorbyt antibody against RBP4 (1:100 in PBST) for 1 h at room temperature in a humidity chamber.

Techniques: Software, Expressing

Journal: Scientific reports

Article Title: Retinol binding protein 4 abundance in plasma and tissues is related to body fat deposition in cattle.

doi: 10.1038/s41598-019-44509-4

Figure Lengend Snippet: Figure 2. Relative protein abundance of RBP4 in different adipose tissues and the liver of high carcass fat (HCF, n = 20) and low carcass fat (LCF, n = 18) bulls at an age of 18 months. Data were normalized to total protein in each lane and are expressed as LSmean ± SE. * and ** indicate significant differences between groups with P < 0.05 and P < 0.01, respectively. A representative blot image of the liver, cropped to the respective band, is shown above the graph. The complete blot and a blot of SCF are shown in Supplemental Fig. 2. SCF – subcutaneous fat, OF – omental fat, PF – perirenal fat, IF – intestinal fat.

Article Snippet: After blocking with 10% goat serum in PBST for 15 min, sections were incubated with the Biorbyt antibody against RBP4 (1:100 in PBST) for 1 h at room temperature in a humidity chamber.

Techniques: Quantitative Proteomics

Journal: Scientific reports

Article Title: Retinol binding protein 4 abundance in plasma and tissues is related to body fat deposition in cattle.

doi: 10.1038/s41598-019-44509-4

Figure Lengend Snippet: Figure 3. Cellular localization of RBP4 in subcutaneous fat (a–c), muscle tissue (d–f), and bovine liver (g–i) by immunohistochemistry. (a,d,g) RBP4 detection (secondary antibody: Alexa Fluor 488 conjugated goat anti rabbit IgG, green), (b,e,h) nuclear staining with Hoechst 33258 (blue), (c,f,i) merged images. Arrows indicate stained cells with cytoplasmic RBP4 localization. Images were equally enhanced in contrast. AD – adipocyte, CT – connective tissue, MF – muscle fibre.

Article Snippet: After blocking with 10% goat serum in PBST for 15 min, sections were incubated with the Biorbyt antibody against RBP4 (1:100 in PBST) for 1 h at room temperature in a humidity chamber.

Techniques: Immunohistochemistry, Staining

Journal: Scientific reports

Article Title: Retinol binding protein 4 abundance in plasma and tissues is related to body fat deposition in cattle.

doi: 10.1038/s41598-019-44509-4

Figure Lengend Snippet: Figure 4. Cellular localization of RBP4 (a, MFP 590 conjugated goat anti rabbit IgG, red) and the macrophage marker CD163 (b, Alexa Fluor 488 conjugated goat anti mouse IgG, green) in bovine liver. (c) Nuclear staining with Hoechst 33258 (blue); (d) merged image. Open arrows indicate co-localization, closed arrows indicate cells exclusively immunoreactive for RBP4, and arrow heads indicate exclusively CD163 immunoreactive macrophages.

Article Snippet: After blocking with 10% goat serum in PBST for 15 min, sections were incubated with the Biorbyt antibody against RBP4 (1:100 in PBST) for 1 h at room temperature in a humidity chamber.

Techniques: Marker, Staining

Journal: Scientific reports

Article Title: Retinol binding protein 4 abundance in plasma and tissues is related to body fat deposition in cattle.

doi: 10.1038/s41598-019-44509-4

Figure Lengend Snippet: Figure 5. Abundance of RBP4 protein in plasma of HCF and LCF bulls at 8 months and 18 months of age. Data are expressed as LSmean ± SE. * and ** indicate significant differences between groups with P < 0.05 and P < 0.01, respectively. HCF – high carcass fat, n = 20; LCF – low carcass fat, n = 18. A representative blot image is shown in Supplemental Fig. 4.

Article Snippet: After blocking with 10% goat serum in PBST for 15 min, sections were incubated with the Biorbyt antibody against RBP4 (1:100 in PBST) for 1 h at room temperature in a humidity chamber.

Techniques: Clinical Proteomics

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have 10- to 6-fold increases in serum RBP4 levels and maintain a normal body mass and fasting blood glucose, serum insulin, and triglyceride levels. (A) Total (mouse and human transgene) RBP4 levels in mouse serum (n ≥ 4 mice per age-genotype combination; WT, wild type). (B) TTR levels in mouse serum at 2 months of age (n ≥ 5 mice per genotype; data were analyzed by Student's t test). (C and D) Blood glucose levels in mice that had fasted for 6 h (n ≥ 6 mice per measurement). (E and F) Body mass measurements in mice that had fasted for 6 h (n ≥ 6 mice per measurement). (G) Insulin levels in the sera of 9-month-old mice that had fasted for 6 h (no significant difference between genotypes was detected on the basis of Student's t test). (H) Triglyceride levels in serum from 4-month-old mice that had fasted for 6 h (no significant difference between genotypes was detected on the basis of Student's t test). (I) Adiponectin levels in serum from 4-month-old mice that had fasted for 6 h (no significant difference between genotypes was detected on the basis of Student's t test). (J and K) Results of insulin tolerance tests in which male mice aged 17 to 21 weeks (n = 7 to 8 mice per genotype) were fasted for 6 h and then given a single i.p. injection of insulin (0.75 unit/kg) at time zero, followed by blood glucose measurements at 0, 15, 30, 60, 90, and 120 min postinjection. All graph values are means ± SEMs. **, P < 0.01 by Student's t test; ***, P < 0.001 by Student's t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Injection

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: Mouse serum levels of retinol and RBP4 a

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have age-dependent inner retinal degeneration. (A and B) Quantitative histological analyses of the total retina, INL, and ONL thickness for retinas from mice aged 1 month (A) or 6 months (B) (WT, wild-type; ONH, optic nerve head). Values are means ± SEMs for ≥8 mice for each data point. *, P < 0.05 by one-way analysis of variance (ANOVA) with Tukey's post hoc test; **, P < 0.01 by one-way ANOVA with Tukey's post hoc test; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test. (C to E) Representative confocal images of retinal paraffin sections from 9-month-old mice immunofluorescently labeled for rhodopsin (C), cone S-opsin (D), cone M-opsin (E). (F) Representative confocal images of retinal cryosections from 6-month-old mice immunofluorescently labeled for cone arrestin. DAPI counterstaining is shown in blue. Confocal images were acquired under a 63× magnification objective, but rhodopsin images are shown with an additional digital zoom of ×2. All images shown represent the maximum stack of z-plane acquisition. All images were acquired from central retina sections and are representative of those from 4 to 6 independent samples from mice of each genotype. Bars, 10 μm (C to E) and 20 µm (F).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Labeling

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: Visual acuity is significantly reduced in RBP4-Tg mice. (A to C) RBP4 and albumin levels in perfused retinas from mice (WT, wild type) aged 6 months (n ≥ 5 mice per genotype); (D and E) OKT response to changes in spatial frequency; (F and G) OKT response to changes in grating contrast. All OKT analyses were performed on ≥6 mice per genotype. Values are means ± SEMs. n.s., not significant by Student's t test; *, P < 0.05 by Student's t test; **, P < 0.01 by Student's t test; ***, P < 0.001 by Student's t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have an age-related loss of rod and cone bipolar cells. (A to G) Representative confocal images of retinal cryosections from 6-month-old mice immunofluorescently labeled for distinct INL cell types, as follows: all bipolar cells (green, Chx10) and rod bipolar cells (red, PKC-α) (A), rod bipolar cells (PKC-α) (B), cone bipolar cells (secretagogin) (C), calcium-binding amacrine and ganglion cells (calretinin) (D), glycinergic amacrine cells (GLYT1) (E), horizontal cells (calbindin) (F), and Müller cells (glutamine synthetase [GS]) (G). (H) Representative confocal images of retinal whole mounts immunolabeled with NeuN (green) to stain retinal ganglion cell bodies and beta-III tubulin (red) to stain ganglion axonal processes. In each panel, the image on the left is from a wild-type mouse retina and the image on the right is from an RBP4-Tg mouse retina. Confocal images were acquired under a 63× (A to G) or 40× (H) magnification objective, but the secretagogin (C) and calbindin (F) images are shown with an additional digital zoom of ×2. All images shown represent the maximum stack of z-plane acquisition. All images were acquired from central retina sections and are representative of those from 3 to 7 independent samples from mice of each genotype. Bars, 10 μm.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Labeling, Binding Assay, Immunolabeling, Staining

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have early-onset retinal gliosis and microglia activation. (A and B) Representative confocal images of retinal cryosections from 1-month-old mice (A) and 6-month-old (B) mice immunofluorescently labeled for GFAP, a marker of gliosis (WT, wild type). DAPI counterstaining is shown in blue. Confocal images were acquired under a 40× magnification objective and represent the maximum stack of z-plane acquisition. All images were acquired from central retina sections and are representative of those from 3 to 5 independent samples from mice of each genotype. Bars, 10 μm. (C and D) Representative confocal images of retinal whole mounts from 1-month-old mice immunofluorescently labeled for microglia marker CD11b. Confocal images were acquired under a 40× magnification objective, and each image represents a single layer of z-plane acquisition. Arrows, microglia cells with enlarged cell bodies characteristic of activated microglia. All images were acquired from the midperiphery and are representative of those from 3 to 6 independent samples from mice of each genotype. Bars, 40 μm. (E and F) Microglia cell bodies were counted in retinal whole mounts under a 40× magnification objective. Each field of view corresponds to a surface area of 0.14 mm2. Microglia were counted in 3 to 5 retinas per genotype. Values are means ± SEMs. **, P < 0.01 by Student's t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Activation Assay, Labeling, Marker

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have dominant and progressive inner retinal dysfunction leading to secondary photoreceptor dysfunction. (A and B) Scotopic ERG a-wave and b-wave amplitudes in mice aged 1 to 9 months (WT, wild type). (C) Photopic ERG b-wave amplitudes in mice aged 1 to 9 months. Values are means ± SEMs for ≥6 mice per age-genotype combination. n.s., not significant by one-way analysis of variance (ANOVA) with Tukey's post hoc test; *, P < 0.05 by one-way ANOVA with Tukey's post hoc test; **, P < 0.01 by one-way ANOVA with Tukey's post hoc test; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test. (D) Representative scotopic ERG responses to increasing light intensities (0.004 to 40 cd · s/m2) in mice aged 6 months. Note the pronounced b-wave decline compared to the more subtle a-wave reduction.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have deficiencies in photoreceptor ribbon synapses and cone bipolar terminals. (A to E) Representative confocal images of retinal cryosections from 1-month-old (A) or 6-month-old (B to E) mice (WT, wild type) immunolabeled for presynaptic photoreceptor ribbon synapses (red, bassoon; green, ribeye) (A and B), photoreceptor ribbon synapse connection to postsynaptic rod bipolar cell dendrites (red, bassoon; green, PKC-α) (C), photoreceptor ribbon synapse connection to postsynaptic horizontal cell dendrites (red, bassoon; green, calbindin) (D), and photoreceptor presynaptic terminals (red, VLGUT1) and cone bipolar axonal and dendritic terminals (green, ZNP-1) (E). Confocal images were acquired under a 63× magnification objective, but panels A to C are shown with an additional digital zoom of ×4, and panel D is shown with an additional zoom of ×6. All images shown represent the maximum stack of z-plane acquisition. DAPI counterstaining is shown in blue. Arrowheads, displaced VGLUT1; arrows, thinning or loss of ZNP-1 labeling. All images were acquired from central retina sections and are representative of those from 4 to 8 independent samples from mice of each genotype. Bars, 10 μm.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Immunolabeling, Labeling

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have normal levels of retinal visual cycle retinoids and bisretinoids A2E and iso-A2E. (A to C) HPLC-based quantification of visual cycle retinoid isomers in eyes from 2- to 11-month-old mice dark adapted for 16 h prior to euthanization. Values are means ± SEMs for 4 mice per genotype-age combination. ns, not significant. P values were determined by Student's t test. (D) HPLC-based quantification of bisretinoid A2E and iso-A2E levels in eyecups from 9-month-old mice reared in cyclic room light. Values are means ± SEMs for 4 samples (4 eyecups per sample from 8 mice total) per genotype; n.s., not significant by Student's t test. (E) Quantitative RT-PCR was performed to measure RARβ mRNA levels in retinas from mice aged 3 months. Values are means ± SEMs for 3 samples per group. ns, not significant by Student's t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have normal retinal stratification and RPE pigmentation and integrity. (A) Representative retinal fundus images of wild-type (WT) and RBP4-Tg mice aged 6 months. (B) Hematoxylin- and eosin-stained retinal sections from mice aged 6 months. Magnifications, ×40; bars, 20 μm. (C) Bright-field images of retinal whole mounts showing normal RPE pigmentation in 6-month-old mice. Magnifications, ×4. (D) Representative confocal images of RPE whole mounts from 6-month-old mice immunofluorescently labeled for ZO-1 to demarcate tight junctions. Confocal images were acquired under a 63× magnification objective and represent the maximum stack of z-plane acquisition. Bars, 20 μm. The images in panels B and D were acquired from central retina sections. All images are representative of those from 4 to 6 independent samples from mice of each genotype.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Staining, Labeling

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have normal retinal vasculature. (A) Fluorescein angiogram images from wild-type (WT) and RBP4-Tg mice aged 6 months. Each image shown is from a different mouse. (B) Epifluorescent images from whole-mount retinas following a retinal leukostasis procedure in which mice were perfused with FITC-concanavalin A to label adherent leukocytes. Arrows, adherent leukocytes. Magnification, ×20. (C) Quantification of retinal leukostasis. Values are means ± SEMs for ≥3 mice (6 eyes) per genotype-age combination. There was no statistically significant difference between genotypes by two-way analysis of variance.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: Microglia projections extend into the outer nuclear layer in RBP4-Tg mice. (A and B) Representative confocal images of retinal whole mounts from 6-month-old mice immunofluorescently labeled for the microglia marker Iba-1. Confocal images were acquired under a 63× magnification objective, and each image represents the z-series of the inner plexus layer (GCL, ganglion cell layer). Numbered boxes denote cells that are shown at higher magnification to the right side of the original image. All images were acquired from the midperiphery and are representative of those from 3 to 5 independent samples from mice of each genotype. Note the enlarged cell bodies in the microglia of RBP4-Tg mouse retinas. Bars, 40 μm. (C and D) Representative 3D confocal images of retinal whole mounts from 6-month-old mice immunofluorescently labeled for CD31 (red) and either Iba-1 (green) (C) or CD11b (green) (D). All images were acquired from the midperiphery and are representative of those from 3 to 5 independent samples from mice of each genotype. Note the microglia extensions into the ONL in the retinas of RBP4-Tg mice only.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Labeling, Marker

Journal: Molecular and Cellular Biology

Article Title: Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

doi: 10.1128/MCB.00181-15

Figure Lengend Snippet: RBP4-Tg mice have retinal neuroinflammation. qRT-PCR analyses showed increased levels of pro-IL-18 (A) and reduced levels of pro-IL-1β (B) mRNA expression in retinas from 6-month-old RBP4-Tg mice. (C) Western blots show no retinal expression of mature IL-1β in RBP4-Tg or wild-type mice at age 3 months. Lane +C, 1 μg recombinant mouse IL-1β as a positive control. The upper band near 45 kDa is β-actin, while the lower band near 15 kDa is mature IL-1β. (D and E) Western blot and densitometry (densitomet) analyses show significantly increased expression of mature IL-18 in retinas from RBP4-Tg mice at age 6 months. Densitometry is from 3 independent experiments consisting of 3 mice per genotype. (F) Serum IL-18 levels measured by ELISA. (G) The enzymatic activity of caspase-1 in lysates of retinas from 6-month-old mice was determined by incubation with the colorimetric substrate YVAD-pNA. (H) qRT-PCR analyses show decreased levels of NLRP3 mRNA expression in retinas from 6-month-old RBP4-Tg mice. Values are means ± SEMs for 4 to 6 mice per genotype. n.s., not significant by Student's t test; *, P < 0.05 by Student's t test; **, P < 0.01 by Student's t test; ***, P < 0.001 by Student's t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse adiponectin (catalog no. MRP300; R&D Systems, Minneapolis, MN), mouse insulin (catalog no. 90080; Crystal Chem, Downers Grove, IL), mouse transthyretin (TTR; catalog no. IRKTAH1161; Innovative Research, Novi, MI), human RBP4 (catalog no. DRB400; R&D Systems, Minneapolis, MN), mouse RBP4 (catalog no. MRB400; R&D Systems, Minneapolis, MN), and mouse IL-18 (catalog no. 7625; MBL International Corp., Woburn, MA) were used to quantify the target molecules in serum samples.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Recombinant, Positive Control, Enzyme-linked Immunosorbent Assay, Activity Assay, Incubation

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: TIPS construction and validation. ( a ): Based on 302 TIDEGs, the training set is screened using univariate Cox analysis to identify 18 prognosis-related genes. Unifactorial analysis of TIDEGs in the training set. ( b ): Three TICPGs are identified using multivariate Cox analysis, including KLRK1 (β=-0.303), LTB (β=-0.562), and RBP4 (β = 0.161). The risk score formula is as follows: risk score=(-0.303×KLRK1)+(-0.562×LTB)+(0.161×RBP4). ( c ): Survival curve analysis of TIPS in the high-risk group vs. low-risk group in the training set, ROC curves, distribution of risk scores, distribution of survival statuses, and heatmap of TICPGs. The area under the curve (AUCs) of TIPS at 1, 3, and 5 years are 0.7, 0.704, and 0.739, respectively. ( d ): Heatmap of TIPS in the test set of high-risk group vs. low-risk group survival curve analysis, ROC curve, risk score distribution, survival status distribution, TICPGs heat map. In the test and full sets ( n = 158), the high- and low-risk groups are divided based on optimal cutoff value; KM curves showed that the prognosis of the low-risk group was significantly better ( p < 0.05). ROC analysis shows the AUCs in the test set at 1, 3, and 5 years of 0.737, 0.64, and 0.647, respectively. ( e ): TIPS in the full set of high-risk group vs. low-risk group survival curve analysis, ROC curve, risk score distribution, survival status distribution, TICPGs heat map. Corresponding AUCs in the full set are 0.713, 0.677, and 0.711, respectively. ( f ): Full set of univariate analysis of TIPS vs. traditional clinical indicators. ( g - i ): ROC curve of the AUC values in TIPS at 1, 3, and 5 years. (j): Full set of multifactorial analyses of TIPS versus STAGE, T versus N staging. DEGs, differentially expressed genes; EMT, epithelial-mesenchymal transition; ROC, receiver operating characteristic; TIPS, TME immune-related genes prognostic signature; TIDEGs, TME immune-related DEGs; TME, tumor microenvironment.

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Biomarker Discovery

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: Effects of RBP4 overexpression or knockdown on TSCC cell proliferation, migration, and invasion. ( a ): Differential analysis of KLRK1, LTB, and RBP4 expression in tumor tissues vs. paired paracancerous normal tissues in the TCGA-TSCC dataset. ( b ): In the GSE13601 ( p = 5e-06), GSE78060 ( p = 0.082), and GSE31056 ( p = 1e-05) datasets, the RBP4 expression in tumor samples vs. paired paracancerous normal tissue samples. RBP4 differential expression analysis in tumor samples relative to paraneoplastic normal tissue samples in GSE13601 , GSE78060 , and GSE31056 datasets. The left panel shows the volcano plots of DEGs; the right panel shows the box plots of RBP4 expression levels in tumor samples versus paraneoplastic normal samples. ( c ): Immunohistochemistry observation of RBP4 expression in TSCC and paraneoplastic tissues. The upper panel shows the expression of RBP4 in TSCC tissues. Scale bar: 200 μm. The lower panel shows the expression of RBP4 in paraneoplastic tissues. Scale bar: 50 μm. ( d - g ): Overexpression or knockdown of RBP4 in CAL27 and SCC-15 cells using lentivirus and validated by qRT-PCR with western blotting experiments. ( h ): CCK-8 method is used to analyze the effect of changes in RBP4 levels on the proliferative ability of TSCC cells. ( i ): In vivo experiments validate the effect of changes in RBP4 expression levels on the TSCC cell proliferation rate. ( j ): Effects of changes in RBP4 expression levels on TSCC cell migration ability were observed by scratch assay. ( k ): Effects of changes in RBP4 expression levels on TSCC migration invasion ability were observed by Transwell assay. DEGs, differentially expressed genes; TCGA, The Cancer Genome Atlas Program; TSCC, tongue squamous cell carcinoma. (*Compared with NC, * p < 0.05, ** p < 0.01, *** p < 0.001,*** p < 0.0001).

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Over Expression, Knockdown, Migration, Expressing, Quantitative Proteomics, Immunohistochemistry, Quantitative RT-PCR, Western Blot, CCK-8 Assay, In Vivo, Wound Healing Assay, Transwell Assay

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: Mechanism study of RBP4 affecting TSCC cell proliferation, migration, and invasion. ( a ): Bioinformatic analysis of the effect of RBP4 on the PI3K/Akt/mTOR signaling pathway. ( b ): Western blotting to detect the effect of changes in the expression level of RBP4 on the activation level of the PI3K/Akt/mTOR pathway in TSCC cells. ( c ): After addition of 740 Y-P (0.2 mM), western blotting detects the alteration of the effect of RBP4 on the PI3K/Akt/mTOR pathway. ( d ): CCK-8 method is used to analyze the changes in the effect of RBP4 on the proliferative capacity of TSCC cells after the addition of 740 Y-P (0.2 mM). ( e - f ): To explore the mechanism by which changes in RBP4 levels affect TSCC cell migration and invasion, we determine the changes in Snail expression in RBP4 overexpressing and RBP4 knockdown TSCC cells. qRT-PCR and western blotting are used to detect the effect of RBP4 on the expression level of Snail and EMT-related proteins. ( g ): PI3K agonist treatment reverses the effects of RBP4 overexpression on Snail/E-cadherin/N-cadherin/Vimentin regulation. Western blotting to detect the changes of the effect of RBP4 on the PI3K/Akt/mTOR pathway after the addition of 740 Y-P (0.2 mM). ( h ): PI3K agonist treatment enhances cell migration and invasion, suggesting that RBP4 suppresses the TSCC malignant phenotype by inhibiting the PI3K/Akt/mTOR-Snail axis. Scratch assay to detect the changes of the effect of RBP4 on the migration ability of TSCC cells after the addition of 740 Y-P (0.2 mM). Changes in the effect of TSCC cell migration ability. ( i ): Transwell assay to detect changes in the effect of RBP4 on TSCC cell invasion ability after addition of 740 Y-P (0.2 mM). TSCC, tongue squamous cell carcinoma. (*Compared with NC, * p < 0.05, ** p < 0.01, *** p < 0.001,*** p < 0.0001).

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Migration, Western Blot, Expressing, Activation Assay, CCK-8 Assay, Knockdown, Quantitative RT-PCR, Over Expression, Wound Healing Assay, Transwell Assay

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: RBP4 in TSCC cells affects macrophage phenotypic transformation. To investigate the association between RBP4 expression levels and tumor-infiltrating macrophage phenotypes in TSCC tissues, we perform a correlation bioconfidence analysis using the GEO database. ( a ): In the GSE13601 dataset, the left graph represents the distribution of macrophage M1 polarization in the subgroup of tumor samples versus paracancerous samples, and the right graph represents the distribution of macrophage M1 polarization in the subgroup of samples with high and low RBP4 expression in tumor tissues. ( b ): In the GSE31056 dataset, the left graph represents the distribution of macrophage M1 polarization in the subgroup of tumor samples versus paracancerous samples, and the right panel represents the distribution of macrophage M1 polarization in the subgroup of samples with high and low tumor tissue RBP4 expression. Significance is calculated using the Wilcoxon rank-sum test. ( c ): CD86 and RBP4 expression levels are determined using immunohistochemical staining of paraneoplastic and TSCC tissues. ( d ): Flow cytometry is performed to detect phenotypic changes in macrophages co-cultured with TSCC. TSCC, tongue squamous cell carcinoma. (*Compared with NC, ** p < 0.01, *** p < 0.001,*** p < 0.0001).

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Transformation Assay, Expressing, Immunohistochemical staining, Staining, Flow Cytometry, Cell Culture

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: RBP4 promotes macrophage M1 polarization by activating the TLR4/NF-κB pathway. ( a ): KEGG Bar Charts of human recombinant RBP4 protein-treated M0 macrophage cells after transcriptome sequencing. ( b ): Correlation analysis of RBP4 and TLR4 protein expression using Spearman and Pearson methods. ( c ): Western blotting assay to detect the effect of human recombinant RBP4 on macrophage TLR4/NF-KB pathway and the change of the effect of RBP4 action after the addition of TAK242. ( d , e ): qRT-PCR and western blotting are used to detect the effect of the human recombinant RBP4 protein on the macrophage phenotype and the effect of RBP4 action after the addition of TAK242. ( f , g ): Western blotting experiments are used to detect the effects of changes in RBP4 expression levels on the macrophage TLR4/NF-κB pathway in a Transwell co-culture system. ( h ): Western blotting experiments are used to detect the effects of tumor-secreted RBP4 on the macrophage TLR4/NF-κB pathway after the addition of TAK242. Changes in activation level. ( i ): Western blotting experiments are used to detect the effect of tumor-secreted RBP4 on macrophage phenotype after addition of TAK242 in Transwell co-culture system. TAK242 treatment blocks RBP4 overexpression induced by TSCC cells in the macrophage TLR4/p-NF-κB upregulation and M1-type polarization. KEGG, Kyoto Encyclopedia of Genes and Genomes; TSCC, tongue squamous cell carcinoma. (*Compared with Control, * p < 0.05, ** p < 0.01, *** p < 0.001,*** p < 0.0001).

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Recombinant, Sequencing, Expressing, Western Blot, Quantitative RT-PCR, Co-Culture Assay, Activation Assay, Over Expression, Control

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: Effect of RBP4 on macrophage phenotype. ( a ): Animals were grouped according to the injection of different cells, and the tumor volume, growth curve, and mass were measured and recorded. ( b ): The levels of CD86 and CD206 cell infiltration in the sections of transplanted tumors from each group are analyzed using immunofluorescence staining. ( c ): Changes in the activation level of the TLR4/NF-KB pathway in each group of transplanted tumor tissues are detected using Western blotting. ( d ): Mechanistic diagram of the effects of changes in RBP4 levels on the proliferation, migration, and invasion abilities of TSCC cells and the effects of tumor-secreted RBP4 on the phenotype of TAMs. TAMs, tumor-associated macrophages; TSCC, tongue squamous cell carcinoma. (*Compared with RAW264.7 + Control, * p < 0.05, ** p < 0.01).

Article Snippet: Transcriptome sequencing showed that RBP4 activated the TLRs/NF-κB pathway (Fig. a) and that TLR4 and RBP4 expression were positively correlated (Spearman, Fig. b).Western blotting showed elevated expression of TLR4 and p-NF-κB in macrophages incorporating the human recombinant RBP4 protein (with no NF-κB changes) (Fig. c).QRT-PCR and Western blotting confirmed that RBP4 significantly up-regulated M1 markers (CD86, iNOS) and inhibited M2 markers (CD206, Arg-1) (Fig. d, e), whereas the TLR4 inhibitor TAK242 (0.2 Mm, HY-11109, MCE) reversed this effect ( Fig. c, e).

Techniques: Injection, Immunofluorescence, Staining, Activation Assay, Western Blot, Migration, Control

Journal: Stem Cell Reports

Article Title: RBP4-STRA6 Pathway Drives Cancer Stem Cell Maintenance and Mediates High-Fat Diet-Induced Colon Carcinogenesis

doi: 10.1016/j.stemcr.2017.06.002

Figure Lengend Snippet: STRA6 and RBP4 Are Upregulated in Colorectal Cancer Patients (A) Kaplan-Meier plot showing differences in disease-free survival percentages between colorectal cancer patients with high or low expression of STRA6 or RBP4. TCGA dataset, available through cBioPortal, was used. H.R., hazard ratio; CI, confidence interval. (B and C) Levels of STRA6 expression in normal versus adenocarcinoma samples (B) and in normal versus rectal cancer patients (C). (D) STRA6 mRNA levels in samples from patients showing complete or partial pathological response to chemoradiation. (E) Data analysis showing levels of RBP4 mRNA in matched samples from primary and liver metastasis of colon cancer. (F) Levels of RBP4 mRNA in rectal cancer patient samples grouped by 3-year recurrence. (G and H) Levels of RBP4 mRNA in colon tumors with high versus low/stable microsatellite instability (MSI) (G) and in tumors with KRAS mutation (MUT) versus the wild-type (WT) (H). (I) RBP4 levels measured in serum of KRAS WT (n = 16) and KRAS mutant (n = 14) patients. Boxes represent the sample range and whiskers are 1 SD from the mean. Squares within the boxes represent mean values. ∗ p < 0.05; n.s., not significant

Article Snippet: Human RBP4 in serum samples of patients was quantified using an RBP4 Quantikine ELISA kit (R&D Systems) following the manufacturer's protocols.

Techniques: Expressing, Mutagenesis

Journal: Stem Cell Reports

Article Title: RBP4-STRA6 Pathway Drives Cancer Stem Cell Maintenance and Mediates High-Fat Diet-Induced Colon Carcinogenesis

doi: 10.1016/j.stemcr.2017.06.002

Figure Lengend Snippet: RBP4-STRA6 Pathway Is Necessary for Expression of CSC Markers (A) Cell lysates from SW480 cells transfected with non-target, STRA6, or RBP4 shRNA were run on SDS-PAGE and immunoblotted with indicated antibodies. (B and C) mRNA levels of STRA6 (B) and RBP4 (C) in the indicated SW480 stable lines. Data are presented as mean ± SD from n = 3 independent experiments ∗ p < 0.05; # p < 0.01. (D) Viability of SW480 cells stably expressing control, STRA6, or RBP4 shRNAs measured in triplicates for 5 days using trypan blue and Countess II FL. Data are presented as mean ± SE. ∗ p < 0.05, significant difference between control and individual cell lines at each time point. (E) Representative immunoblots of total and cleaved caspase-3 on cells in (A) treated with 10 μM etoposide for 72 hr. (F and G) Representative immunoblots from three independent experiments showing levels of SOX2 and NANOG in SW480 stable lines. Cells were grown in delipidated medium for 18 hr before harvesting. Actin was used as loading control.

Article Snippet: Human RBP4 in serum samples of patients was quantified using an RBP4 Quantikine ELISA kit (R&D Systems) following the manufacturer's protocols.

Techniques: Expressing, Transfection, shRNA, SDS Page, Stable Transfection, Control, Western Blot

Journal: Stem Cell Reports

Article Title: RBP4-STRA6 Pathway Drives Cancer Stem Cell Maintenance and Mediates High-Fat Diet-Induced Colon Carcinogenesis

doi: 10.1016/j.stemcr.2017.06.002

Figure Lengend Snippet: RBP4 Drives Tumor Survival of SW480 Cells (A) Tumor growth in athymic NCr mice injected with SW480 cells (5 × 10 6 ) stably expressing non-target or RBP4 shRNA. Data for control tumors are mean ± SE (n = 8 tumors). (B) Tumor volumes of individual tumor at 15 days after injection are plotted. Mean (n = 8 tumors) and SE for the control are shown. (C) Representative tumors are shown that originate from SW480 cells stably expressing control or RBP4 shRNA. (D) Average tumor volume at experimental endpoint and number of tumors initiated in each group are shown in the table. (E–G) Levels of RBP4 (E), SOX2 (F), and LGR5 (G) mRNAs in SW480 tumors. Mean and SE for the control samples (n = 4 tumors) are shown. (H) Immunoblots showing levels of SOX2 and GAPDH. (I) Immunoblot showing levels of p-STAT3 and total STAT3 in SW480. (J) Immunoblots in (I) were quantified using ImageJ software, and fold changes are presented. Mean and SEs for the control samples (n = 4 tumors) are shown.

Article Snippet: Human RBP4 in serum samples of patients was quantified using an RBP4 Quantikine ELISA kit (R&D Systems) following the manufacturer's protocols.

Techniques: Injection, Stable Transfection, Expressing, shRNA, Control, Western Blot, Software

Journal: Stem Cell Reports

Article Title: RBP4-STRA6 Pathway Drives Cancer Stem Cell Maintenance and Mediates High-Fat Diet-Induced Colon Carcinogenesis

doi: 10.1016/j.stemcr.2017.06.002

Figure Lengend Snippet: STRA6 and RBP4 Maintain CSC Frequency (A) Low-magnification images of SW480 cells grown as non-adherent spheres. Loss of sphere formation ability upon STRA6 or RBP4 knockdown is shown. (B) (Upper panel) Sphere-initiating cell frequency from an in vitro limiting dilution assay was calculated using ELDA software. A representative frequency estimate calculated from 24 biological replicates of each cell dose is shown. Error bars represent 95% confidence intervals. ∗ p < 0.05. (Lower panel) Stem cell frequency estimates within confidence intervals and fold-change differences between control and designated lines. (C) (Upper panel) SW480 cells stably transfected with STRA6 or RBP4 shRNA were injected at limiting doses into NOD-SCID gamma mice (n = 5–6 for each dose) and the ability to initiate tumor formation was evaluated. Plots show tumor-initiating cell frequency calculated using ELDA software. Error bars represent 95% confidence intervals. ∗ p < 0.05. (Lower panel) Stem cell frequency estimates within confidence intervals and fold changes between control and indicated lines.

Article Snippet: Human RBP4 in serum samples of patients was quantified using an RBP4 Quantikine ELISA kit (R&D Systems) following the manufacturer's protocols.

Techniques: Knockdown, In Vitro, Limiting Dilution Assay, Software, Control, Stable Transfection, Transfection, shRNA, Injection

Journal: Stem Cell Reports

Article Title: RBP4-STRA6 Pathway Drives Cancer Stem Cell Maintenance and Mediates High-Fat Diet-Induced Colon Carcinogenesis

doi: 10.1016/j.stemcr.2017.06.002

Figure Lengend Snippet: RBP4-STRA6 Pathway Maintains CSC Frequency in a PDX Model (A) Sphere-initiating cell frequency of PDX 656 cells stably transfected with control or STRA6 (left) or RBP4 (right) shRNAs. A representative frequency estimate calculated from 24 biological replicates of each cell dose is shown. Error bars represent 95% confidence intervals. ∗ p < 0.05. Stem cell frequency estimates within confidence intervals and fold differences between control and designated lines are shown in the table on the far right. (B) Distribution of CD44-negative and -positive cells upon stable knockdown of STRA6 (top) or RBP4 (bottom) in PDX 656. FITC-immunoglobulin G (IgG) isotype control was used to set up the gates. Percentage of cells in each fraction is shown.

Article Snippet: Human RBP4 in serum samples of patients was quantified using an RBP4 Quantikine ELISA kit (R&D Systems) following the manufacturer's protocols.

Techniques: Stable Transfection, Transfection, Control, Knockdown

Journal: Stem Cell Reports

Article Title: RBP4-STRA6 Pathway Drives Cancer Stem Cell Maintenance and Mediates High-Fat Diet-Induced Colon Carcinogenesis

doi: 10.1016/j.stemcr.2017.06.002

Figure Lengend Snippet: HFD Increases STRA6 and LGR5 in a Xenograft Model (A) Body weight of NCr nude mice fed either a regular rodent chow (RD) or a high-fat/high-sucrose diet (HFD) for 18 weeks. Arrow indicates the time point (15 weeks) at which cells were injected. Inset: immunoblot of RBP4 in serum of RD- or HFD-fed mice after 15 weeks. Data are mean ± SEM (n = 10 mice). (B) NCr male mice in (A) were injected with 5 × 10 6 SW480 cells stably expressing GFP shRNA or STRA6 shRNA after 15 weeks on HFD. Tumor growth at both injected sites was monitored twice a week. Data are mean ± SEM (n = 7–9 tumors). # p < 0.01 (C) Tumor volumes at endpoint of the experiment in (A). Data are mean ± SEM (n = 7–9 tumors). ∗ p < 0.05; # p < 0.01. Fold changes in tumor volume between different conditions and their statistical analysis is shown in the table on the right. (D) (Top) Immunoblot for STRA6 in tumors at endpoint in (A). (Bottom) Quantification of the immunoblot. Data are mean ± SEM (n = 3 tumors). ∗∗ p < 0.05. (E and F) Expression levels of SOX2 (E) and LGR5 (F) in tumors at endpoint in (B). Data are mean ± SEM (n = 3–5 tumors). ∗ p < 0.01; ∗∗ p < 0.05. (G) Immunoblot for pSTAT3 in tumors arising from SW480 stable lines in mice fed an HFD (top) and the quantification of the immunoblot (bottom). Data are mean ± SEM (n = 3 tumors). # p < 0.01 (H) Expression levels of STAT3 target genes in the same tumors in (G) from mice fed an HFD. Data are mean ± SD (n = 3 tumors). # p < 0.01.

Article Snippet: Human RBP4 in serum samples of patients was quantified using an RBP4 Quantikine ELISA kit (R&D Systems) following the manufacturer's protocols.

Techniques: Injection, Western Blot, Stable Transfection, Expressing, shRNA

Journal: Communications Biology

Article Title: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

doi: 10.1038/s42003-024-07052-1

Figure Lengend Snippet: Immunoblotting analysis of RBP4 protein levels and PCV2 capsid protein (Cap) expression in 3D4/21 cells and PK-15 cells infected with PCV2 (MOI = 0.2, the same dose below) at the indicated periods ( a ) or infected with PCV2 with increased dose for 36 h ( b ). Quantitative real-time PCR (qPCR) analysis of RBP4 mRNA expression in 3D4/21 cells and PK-15 cells infected with PCV2 at the indicated periods ( c ) or infected with PCV2 with increased dose for 36 h ( d ). e Immunoblotting analysis of RBP4 protein expression in 3D4/21 cells and PK-15 cells left untreated or infected with PCV2 for 36 h following treatment with cycloheximide (CHX, 50 μM) for the indicated periods. Densitometric quantitation of RBP4 was normalized relative to the levels at 0 h conditions. f Immunoblotting analysis of RBP4, phosphorylated (p-) and total p38, p-JNK and JNK, p-p65 and p65, and p-ERK1/2 and ERK1/2 in whole lysates of in 3D4/21 cells or PK-15 cells pretreated with DMSO or p38 inhibitor SB203580 (SB, 10 μM), JNK inhibitor SP600125 (SP, 10 μM), NF-κB inhibitor BAY11 (10 μM), or ERK inhibitor U0126 (10 μM) for 3 h followed by PCV2 infection for 36 h. g Immunoblotting analysis of RBP4, phosphorylated (p-) and total eIF4E, p-p38 and p38, and p-ERK1/2 and ERK1/2 in whole lysates of 3D4/21 cells and PK-15 cells left untreated or infected with PCV2 for the indicated periods. h Immunoblotting analysis of RBP4, p-eIF4E, and total eIF4E in whole-cell lysates of 3D4/21 cells and PK-15 cells left untreated or pretreated with DMSO, U0126 (10 μM), or SB (10 μM) for 3 h followed by PCV2 infection for the indicated periods. Data are representative of three independent experiments ( a , b , e – h ) or pooled from three independent experiments ( c , d , mean ± SD).

Article Snippet: Recombinant human RBP4 (rhRBP4, 3378-LC) was obtained from R&D Systems.

Techniques: Western Blot, Expressing, Infection, Real-time Polymerase Chain Reaction, Quantitation Assay

Journal: Communications Biology

Article Title: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

doi: 10.1038/s42003-024-07052-1

Figure Lengend Snippet: a Immunoblotting analysis of RBP4 and PCV2 ORF protein expression in whole-cell lysates of 3D4/21 cells and PK-15 cells transfected with empty vector (EV) or PCV2 ORF (ORF1–ORF5)-expressing plasmids for 24 h. b Immunoblotting analysis of RBP4 protein levels in whole-cell lysates of 3D4/21 cells and PK-15 cells transfected with EV or PCV2 ORF1-expressing plasmid with increased dose for 24 h. c qPCR analysis of RBP4 mRNA levels in 3D4/21 cells and PK-15 cells transfected with EV or PCV2 ORF1 plasmid for the indicated periods. d Immunoblotting analysis of RBP4, p-eIF4E, and total eIF4E in whole-cell lysates of 3D4/21 cells and PK-15 cells transfected with EV or PCV2 ORF1-5 plasmids for 24 h. e Immunoblotting analysis of RBP4, p-eIF4E and total eIF4E, p-p38 and total p38, and p-ERK1/2 and total ERK1/2 in whole-cell lysates of 3D4/21 cells and PK-15 cells transfected with EV or PCV2 ORF1 plasmid for the indicated periods. Data are representative of three independent experiments ( a , b , d , and e ) or pooled from three independent experiments ( c , mean ± SD).

Article Snippet: Recombinant human RBP4 (rhRBP4, 3378-LC) was obtained from R&D Systems.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Communications Biology

Article Title: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

doi: 10.1038/s42003-024-07052-1

Figure Lengend Snippet: Immunoblotting analysis of PCV2 Cap protein levels ( a ) or TCID 50 assay of viral titers ( b ) in 3D4/21 cells (left panel) or PK-15 (right panel) cells transfected with empty vector or Flag-RBP4 plasmid for 12 h followed by PCV2 infection for the indicated periods or 48 h. Immunoblotting analysis of PCV2 Cap protein levels ( c ) or TCID 50 assay of viral titers ( d ) in wild-type (WT) and RBP4-deficient (RBP4-KO) 3D4/21 cells (left panel), WT and RBP4-KO PK-15 cells (right panel) infected with PCV2 for indicated periods or 48 h. Immunoblotting analysis of PCV2 Cap protein levels ( e ) or TCID 50 assay of viral titers ( f ) in RBP4-KO 3D4/21 cells (left panel) and RBP4-KO PK-15 cells (right panel) infected with PCV2 for the indicated periods or 48 h. After 6 h of PCV2 infection, cells were treated with or without recombinant porcine RBP4 (rpRBP4, 30 μg/mL). g Immunoblotting analysis of PCV2 Cap protein levels (left panel) or TCID 50 assay of viral titers (right panel) in primary PAMs infected with PCV2 for the indicated periods or 48 h. After 6 h of PCV2 infection, the cells were treated with or without recombinant porcine RBP4 (rpRBP4, 30 μg/mL). h Immunoblotting analysis of PCV2 Cap protein levels (left panel) or TCID 50 assay of viral titers (right panel) in WT or RBP4-KO BMDMs infected with PCV2 for the indicated periods or 48 h. i Hematoxylin and eosin (H&E) staining of lung (left) and liver (right) sections from mice infected with PCV2 (5 × 10 5 pfu/mouse) for 7 days. Scale bar, 20 μm. Original magnification, ×40. j Histological scores of lung and liver from mice infected by PCV2 as in ( i ). Each symbol represents an individual mouse ( n = 6/group). k Viral titers in the lung (left) and liver (right) from WT and RBP4-KO mice ( n = 6/group) infected with PCV2 in ( i ). Data are pooled from three independent experiments ( b , d , f and g , h right, j , k , mean ± SD) or representative of three independent experiments ( a , c , e and g , h left, i ). * p < 0.05, ** p < 0.01 (Student’s t test).

Article Snippet: Recombinant human RBP4 (rhRBP4, 3378-LC) was obtained from R&D Systems.

Techniques: Western Blot, Transfection, Plasmid Preparation, Infection, Recombinant, Staining

Journal: Communications Biology

Article Title: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

doi: 10.1038/s42003-024-07052-1

Figure Lengend Snippet: a Immunoblotting analysis of Myc-tagged protein expression of PCV2 ORF1 (left), ORF2 (middle), or ORF3 (right) in whole-cell lysates of WT and RBP4-KO 3D4/21 cells transfected with the expression plasmids for the indicated periods, respectively. b Immunoblotting analysis of PCV2 ORF1 protein levels in whole-cell lysates of WT and RBP4-KO 3D4/21 cells transfected with PCV2 ORF1 expression plasmid for 24 h following treatment with CHX alone (left) (50 μM), CHX together with MG-132 (middle) (30 μM), or CHX together with CQ (right) (20 μM) for the indicated times. Densitometric quantitation of PCV2 ORF1 was normalized relative to the levels of 0 h conditions ( b , lower). c Immunoblotting analysis of PCV2 ORF1 protein expression in whole-cell lysates of RBP4-KO 3D4/21 cells transfected with the PCV2 ORF1 expression plasmid for 24 h and then left untreated or stimulated with rpRBP4 protein (30 μg/mL) for 1 h following treatment with CHX alone, CHX together with MG132, or CHX together with CQ for the indicated times. Densitometric quantitation of PCV2 ORF1 was normalized relative to the levels at 0 h conditions ( c , lower). Immunoblotting analysis of PCV2 ORF1 protein expression in 3D4/21 cells transfected with PCV2 ORF1 expression plasmid for 6 h following treatment with CQ (20 μM) ( d ) or rapamycin (1 μM) ( e ) for the indicated periods. f Immunoblotting analysis of LC3 expression in RBP4-KO 3D4/21 cells stimulated with rpRBP4 with increased dose for 6 h (upper) or stimulated with rpRBP4 protein (30 μg/mL) for indicated periods (lower). Data are representative of three ( a , d , e ) or two ( b , c , f ) independent experiments.

Article Snippet: Recombinant human RBP4 (rhRBP4, 3378-LC) was obtained from R&D Systems.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Quantitation Assay

Journal: Communications Biology

Article Title: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

doi: 10.1038/s42003-024-07052-1

Figure Lengend Snippet: a Immunoprecipitation analysis of WT and RBP4-KO 3D4/21 cells transfected with PCV2 ORF1 and His-Ubiquitin (His-Ub) or His-Ub at K6 (His-Ub-K6), K11 (His-Ub-K11), K27 (His-Ub-K27), K29 (His-Ub-K29), K33 (His-Ub-K33), K48 (His-Ub-K48) or K63 (His-Ub-K63) only for 24 h. b Immunoprecipitation analysis of RBP4-KO 3D4/21 cells expressing PCV2 ORF1 and His-Ub, His-Ub-K48, His-Ub-K63, His-Ub-K48R, or His-Ub-K63R as indicated. Cells were left untreated or stimulated with rpRBP4 protein for 6 h before harvest. c Immunoprecipitation analysis of 3D4/21 cells expressing GFP-TRAF6 together with empty vector (EV), PCV2 ORF1, PCV3 ORF1, or PCV4 ORF1 as indicated. Anti-GFP immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody. Levels of the transfected proteins were analyzed by immunoblotting with anti-Myc and anti-GFP antibodies. d Colocalization of exogenous TRAF6 and PCV2 ORF1 in 3D4/21 cells. Cells were transfected with GFP-TRAF6 and PCV2 ORF1 for 24 h before confocal microscopy. Scale bar, 10 μm. e GST pull-down analysis of the interaction between His-ORF1 and GST, GST-SQSTM1/p62, GST-LC3, or GST-TRAF6 as indicated. Recombinant proteins were pulled down by GST magnetic beads and were analyzed by immunoblotting with anti-His or anti-GST antibodies (upper). Recombinant proteins in the assay were examined by SDS–PAGE and coomassie blue staining (lower). f Schematic drawings of the TRAF6-binding motif Pro-X-Glu-X-X-Ar/Ac. The presence of the putative Pro-X-Glu-X-X-Ar/Ac motifs in ORF1 of PCV2 and other representative circoviruses (PCV3, KX778720.1(Genebank number); PCV4, MK986820.1; PCV1, U49186.1; HuCV2, ON226770.2; GoCV, MT831941.1; DuCV, MN078101.1; and CaCV, JQ821392.1) were analyzed and consistent amino acids are indicated in color. g Immunoprecipitation analysis of the association of PCV2 ORF1 and TRAF6 in 3D4/21 cells transfected with GFP-TRAF6 and wild-type PCV2 ORF1 (WT), or PCV2 ORF1 mutants (P309T, E311A, or P309T/E311A). Anti-Myc immunoprecipitates were analyzed by immunoblotting with anti-GFP or anti-Myc antibody as indicated. Levels of the transfected proteins were analyzed by immunoblotting with anti-GFP or anti-Myc antibody. h Immunoprecipitation analysis of 3D4/21 cells expressing PCV2 ORF1 and His-Ub, His-Ub-K6, His-Ub-K11, His-Ub-K27, His-Ub-K29, His-Ub-K33, His-Ub-K48 or His-Ub-K63 together with or without GFP-TRAF6 as indicated. i Immunoblotting analysis of the indicated proteins in immunoprecipitated samples and whole-cell lysates of RBP4-KO 3D4/21 cells transfected with TRFA6 siRNA or control siRNA (100 nM). Twenty-four hours after transfection, cells were further transfected with PCV2 ORF1 and His-Ub (left) or His-Ub-K63 (right) for 24 h. Cells were then stimulated with rpRBP4 (30 μg/mL) for 6 h before analysis. Data are representative of three ( c – g ) or two ( a , b , h , i ) independent experiments.

Article Snippet: Recombinant human RBP4 (rhRBP4, 3378-LC) was obtained from R&D Systems.

Techniques: Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Western Blot, Confocal Microscopy, Recombinant, Magnetic Beads, SDS Page, Staining, Binding Assay, Control

Journal: Communications Biology

Article Title: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

doi: 10.1038/s42003-024-07052-1

Figure Lengend Snippet: Immunoblotting analysis of PCV2 Cap protein level ( a ) or qPCR analysis of PCV2 DNA copies ( b , left) or TCID50 assay of viral titers ( b , right) in RBP4-KO 3D4/21 cells transfected with empty vector (EV) or Flag-pRBP4 plasmid for 6 h, then cells were treated for 6 h with or without TLR4-specific inhibitor TAK242 (1 μM), followed by infection with PCV2 for the indicated periods or 48 h. Numbers (lower) indicate the grayscale analysis on the protein bands of Cap and RBP4. c Immunoprecipitation analysis of RBP4-KO 3D4/21 cells expressing PCV2 ORF1 and His-Ub-K63 together with or without Flag-RBP4 as indicated for 6 h. Cells were untreated or treated with TAK242 (1 μM) for 18 h before harvest. Data are representative of three independent experiments ( a , c ) or pooled from three independent experiments ( b , mean ± SD). * p < 0.05, ** p < 0.01 (Student’s t test).

Article Snippet: Recombinant human RBP4 (rhRBP4, 3378-LC) was obtained from R&D Systems.

Techniques: Western Blot, TCID50 Assay, Transfection, Plasmid Preparation, Infection, Immunoprecipitation, Expressing