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: Journal of Electrophoresis

Article Title: The usefulness of electrophoretic analysis of urinary retinol-binding protein and retinol-binding protein 4 in type 2 diabetes mellitus patients

doi: 10.2198/jelectroph.53.7

Figure Lengend Snippet: Fig. 2. Western blot profiles of RBP, RBP4 and TTR in urine.

Article Snippet: After blocking, the membrane was washed three times with washing buffer (10 mmol/L Tris, 0.9% NaCl, and 0.05% Tween 20, pH 7.5), and then the membrane was incubated with rabbit anti human RBP polyclonal antibody, prealbumin antibody (Dako Cytomation, Denmark A/S), or goat anti recombinant human RBP4 polyclonal antibody (R&D Systems, Inc. USA) for 1 hr.

Techniques: Western Blot

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 was performed by Genechem, based on the Illumina sequencing platform for transcriptome sequencing (RNA-Seq) of a recombinant human RBP4 (2 μg/ml, HY- P70471 , MCE) protein-treated M0 macrophage cell model. A KEGG pathway enrichment analysis was performed using the R software “clusterProfiler” package after obtaining the differential genes based on gene expression analysis.

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 was performed by Genechem, based on the Illumina sequencing platform for transcriptome sequencing (RNA-Seq) of a recombinant human RBP4 (2 μg/ml, HY- P70471 , MCE) protein-treated M0 macrophage cell model. A KEGG pathway enrichment analysis was performed using the R software “clusterProfiler” package after obtaining the differential genes based on gene expression analysis.

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 was performed by Genechem, based on the Illumina sequencing platform for transcriptome sequencing (RNA-Seq) of a recombinant human RBP4 (2 μg/ml, HY- P70471 , MCE) protein-treated M0 macrophage cell model. A KEGG pathway enrichment analysis was performed using the R software “clusterProfiler” package after obtaining the differential genes based on gene expression analysis.

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 was performed by Genechem, based on the Illumina sequencing platform for transcriptome sequencing (RNA-Seq) of a recombinant human RBP4 (2 μg/ml, HY- P70471 , MCE) protein-treated M0 macrophage cell model. A KEGG pathway enrichment analysis was performed using the R software “clusterProfiler” package after obtaining the differential genes based on gene expression analysis.

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 was performed by Genechem, based on the Illumina sequencing platform for transcriptome sequencing (RNA-Seq) of a recombinant human RBP4 (2 μg/ml, HY- P70471 , MCE) protein-treated M0 macrophage cell model. A KEGG pathway enrichment analysis was performed using the R software “clusterProfiler” package after obtaining the differential genes based on gene expression analysis.

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 was performed by Genechem, based on the Illumina sequencing platform for transcriptome sequencing (RNA-Seq) of a recombinant human RBP4 (2 μg/ml, HY- P70471 , MCE) protein-treated M0 macrophage cell model. A KEGG pathway enrichment analysis was performed using the R software “clusterProfiler” package after obtaining the differential genes based on gene expression analysis.

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

Journal: World Journal of Diabetes

Article Title: Elevated retinol binding protein 4 levels are associated with atherosclerosis in diabetic rats via JAK2/STAT3 signaling pathway

doi: 10.4239/wjd.v12.i4.466

Figure Lengend Snippet: Primer sequences of target genes and internal reference genes

Article Snippet: At 4 °C, the membrane was simultaneously exposed overnight to solution containing primary antibodies [1:300, anti-JAK2 (Bioss, China); 1:5000, anti-p-JAK2 (abcam, United States); 1:1000, anti-STAT3 (Bioss, China); 1:10000, anti-p-STAT3 (abcam, United States); 1:300, anti-Cyclin D1 (Bioss, China); 1:1000, anti- Bcl-2 (Bioss, China); 1:1000, anti-RBP4 (Bioss, China)].

Techniques:

Journal: World Journal of Diabetes

Article Title: Elevated retinol binding protein 4 levels are associated with atherosclerosis in diabetic rats via JAK2/STAT3 signaling pathway

doi: 10.4239/wjd.v12.i4.466

Figure Lengend Snippet: Comparison of indicators among each group

Article Snippet: At 4 °C, the membrane was simultaneously exposed overnight to solution containing primary antibodies [1:300, anti-JAK2 (Bioss, China); 1:5000, anti-p-JAK2 (abcam, United States); 1:1000, anti-STAT3 (Bioss, China); 1:10000, anti-p-STAT3 (abcam, United States); 1:300, anti-Cyclin D1 (Bioss, China); 1:1000, anti- Bcl-2 (Bioss, China); 1:1000, anti-RBP4 (Bioss, China)].

Techniques:

Journal: World Journal of Diabetes

Article Title: Elevated retinol binding protein 4 levels are associated with atherosclerosis in diabetic rats via JAK2/STAT3 signaling pathway

doi: 10.4239/wjd.v12.i4.466

Figure Lengend Snippet: Comparisons of retinol binding protein 4 mRNA expression in each group. a P < 0.05 vs control group, b P < 0.05 vs diabetic rat group. RBP4: Retinol binding protein 4; NC: Control group; DM: Diabetic rat group; DA: Diabetic atherosclerotic rat group.

Article Snippet: At 4 °C, the membrane was simultaneously exposed overnight to solution containing primary antibodies [1:300, anti-JAK2 (Bioss, China); 1:5000, anti-p-JAK2 (abcam, United States); 1:1000, anti-STAT3 (Bioss, China); 1:10000, anti-p-STAT3 (abcam, United States); 1:300, anti-Cyclin D1 (Bioss, China); 1:1000, anti- Bcl-2 (Bioss, China); 1:1000, anti-RBP4 (Bioss, China)].

Techniques: Binding Assay, Expressing

Journal: World Journal of Diabetes

Article Title: Elevated retinol binding protein 4 levels are associated with atherosclerosis in diabetic rats via JAK2/STAT3 signaling pathway

doi: 10.4239/wjd.v12.i4.466

Figure Lengend Snippet: Comparisons of protein expression of retinol binding protein 4 in each group. a P < 0.05 vs control group, b P < 0.05 vs diabetic rat group. RBP4: Retinol binding protein 4; NC: Control group; DM: Diabetic rat group; DA: Diabetic atherosclerotic rat group.

Article Snippet: At 4 °C, the membrane was simultaneously exposed overnight to solution containing primary antibodies [1:300, anti-JAK2 (Bioss, China); 1:5000, anti-p-JAK2 (abcam, United States); 1:1000, anti-STAT3 (Bioss, China); 1:10000, anti-p-STAT3 (abcam, United States); 1:300, anti-Cyclin D1 (Bioss, China); 1:1000, anti- Bcl-2 (Bioss, China); 1:1000, anti-RBP4 (Bioss, China)].

Techniques: Expressing, Binding Assay

Journal: World Journal of Diabetes

Article Title: Elevated retinol binding protein 4 levels are associated with atherosclerosis in diabetic rats via JAK2/STAT3 signaling pathway

doi: 10.4239/wjd.v12.i4.466

Figure Lengend Snippet: Multivariable logistic regression in type 2 diabetes groups ( n = 50)

Article Snippet: At 4 °C, the membrane was simultaneously exposed overnight to solution containing primary antibodies [1:300, anti-JAK2 (Bioss, China); 1:5000, anti-p-JAK2 (abcam, United States); 1:1000, anti-STAT3 (Bioss, China); 1:10000, anti-p-STAT3 (abcam, United States); 1:300, anti-Cyclin D1 (Bioss, China); 1:1000, anti- Bcl-2 (Bioss, China); 1:1000, anti-RBP4 (Bioss, China)].

Techniques: