Journal: Annals of clinical and translational neurology

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

doi: 10.1002/acn3.51890

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

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

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: 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, Ubiquitin Proteomics, 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

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: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Gastric cancer-derived LBP promotes liver metastasis by driving intrahepatic fibrotic pre-metastatic niche formation

doi: 10.1186/s13046-023-02833-8

Figure Lengend Snippet: LBP secretion is associated with GC metastasis to liver and correlated with worse prognosis. A Heatmap of 49 dysregulated secreted proteins detected by DIA-MS secretomic analysis of serum samples from five patients with GC-LM and five patients with GC-NLM. B Clustered heatmap analysis of the top 200 dysregulated mRNAs of 24 pairs of GC tissues and matched normal gastric tissues (|foldchange|> 2; p < 0.05). C Serological levels of LBP and RBP4 were detected by ELISA in the serum of 30 patients with GC-LM, 30 patients with stage II/III GC and 20 healthy volunteers. D WB verified the expression levels of LBP in GC tissues and matched adjacent normal gastric tissues. E Representative IHC images of LBP in paired primary tumours and LM from the same GC patients. Scale bar, 100 μm. F Comparisons of LBP expression in primary GC tumors and LM based on IHC results. G ROC curve analysis was applied based on the previous serological LBP levels of 30 GC-LM patients and 30 GC-NLM patients. H Liver metastasis-free survival analysis of an additional cohort of GC patients based on serological LBP levels. The cut-off value was determined according to the ROC curve. Data are representative of three independent experiments. Data are shown as mean ± SEM, and p values were determined by one-way ANOVA test ( C ) or log rank test ( H ) (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: ELISA kits were used to determine the levels of human RBP4 (KE00056, Proteintech, China), human LBP (KE00134, Proteintech, China), human TGF-β1 (BMS249-4, Thermo Fisher Scientific, USA) in serum samples or cell culture supernatants, following the manufacturer’s instruction.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing