Journal: Regenerative Biomaterials

Article Title: AI-guided design and optimization of a novel KIM-1-targeted peptide for bFGF delivery in acute kidney injury repair

doi: 10.1093/rb/rbag050

Figure Lengend Snippet: Construction and bioactivity evaluation of WEV-bFGF. ( A ) Spatial structure prediction of WEV-bFGF recombinant protein and natural bFGF; ( B ) SDS-PAGE of bFGF, KIT-bFGF and WEV-bFGF; ( C ) Western blot of native bFGF, KIT-bFGF and WEV-bFGF; ( D ) The biological activities of native bFGF, KIT-bFGF and WEV-bFGF by CCK-8 assay; ( E ) The fluorescence distribution of Delight 800 labeled recombinant proteins in hypoxic HK-2 cells; ( F ) Statistical analysis of fluorescence intensity of Dylight 800 in hypoxic HK-2 cells; Scale bar = 25 μm; ( G ) The protective effects of recombinant proteins on HK-2 cells after hypoxic injury by the CCK8 assay. All quantitative data are presented as mean ± SD, * P < 0.05, ** P < 0.01.

Article Snippet: Subsequently, in accordance with the instructions of the Human bFGF ELISA Kit (EK0441, Boster, China), the protein extracted from kidney tissue and the content of bFGF in serum were determined.

Techniques: Recombinant, SDS Page, Western Blot, CCK-8 Assay, Fluorescence, Labeling

Journal: Regenerative Biomaterials

Article Title: AI-guided design and optimization of a novel KIM-1-targeted peptide for bFGF delivery in acute kidney injury repair

doi: 10.1093/rb/rbag050

Figure Lengend Snippet: The evaluation of targeting capacity of WEV-bFGF in ischemic kidney in vivo . ( A ) At 6 h after administration, the in vivo fluorescence distribution of Dylight800-labeled recombinant proteins in major organs of AKI-injured rats by the small animal imaging system; ( B ) Western blot showing bFGF protein expression in ischemic renal tissue 6 h after administration; ( C ) Quantitative analysis of relative bFGF protein expression at 6 h; ( D ) The content of bFGF in ischemic kidneys by ELISA assay at 6 h after administration; ( E ) The content of bFGF in serum at 6 h after administration; ( F ) Immunofluorescence colocalization of bFGF and KIM-1 in the frozen sections at 6 h after administration, Scale bar = 50 μm; ( G ) Colocalization analysis of PBS, bFGF, KIT-bFGF and WEV-bFGF groups at 6 h after administration; ( H ) At 24 h after administration, the in vivo fluorescence distribution of Dylight800-labeled recombinant proteins in major organs of AKI-injured rats by the small animal imaging system; ( I ) Western blot showing bFGF protein expression in ischemic renal tissue 24 h after administration; ( J ) Quantitative analysis of relative bFGF protein expression at 24 h; ( K ) The content of bFGF in ischemic kidneys by ELISA assay at 24 h after administration; ( L ) The content of bFGF in serum at 24 h after administration; ( M ) Immunofluorescence staining showing colocalization of bFGF and KIM-1 at 24 h after administration, scale bar = 50 μm; ( N ) Colocalization analysis of PBS, bFGF, KIT-bFGF and WEV-bFGF groups at 24 h after administration. All quantitative data are presented as mean ± SD, * P < 0.05, ** P < 0.01, N = 8.

Article Snippet: Subsequently, in accordance with the instructions of the Human bFGF ELISA Kit (EK0441, Boster, China), the protein extracted from kidney tissue and the content of bFGF in serum were determined.

Techniques: In Vivo, Fluorescence, Labeling, Recombinant, Imaging, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Regenerative Biomaterials

Article Title: AI-guided design and optimization of a novel KIM-1-targeted peptide for bFGF delivery in acute kidney injury repair

doi: 10.1093/rb/rbag050

Figure Lengend Snippet: Functional tests and morphological analysis of the kidneys. ( A ) Experimental design of WEV-bFGF for the treatment of I/R; ( B ) Statistical analysis of serum Scr content for renal function evaluation at 24 h after renal I/R injury in rats; ( C ) Statistical analysis of serum Scr content for renal function evaluation at 24 h after renal I/R injury in rats; ( D ) H&E staining for kidney histopathological evaluation after I/R injury. Arrows indicated the sites of renal tubular injury. Scale bar = 20 μm; ( E ) Statistical analysis of renal tubular injury score; ( F ) MASSON staining for renal tissue fibrosis evaluation after I/R injury. Scale bar = 50 μm; ( G ) Statistical analysis of renal tissue fibrosis. All quantitative data are presented as mean ± SD, * P < 0.05, ** P < 0.01, N = 8.

Article Snippet: Subsequently, in accordance with the instructions of the Human bFGF ELISA Kit (EK0441, Boster, China), the protein extracted from kidney tissue and the content of bFGF in serum were determined.

Techniques: Functional Assay, Staining

Journal: Regenerative Biomaterials

Article Title: AI-guided design and optimization of a novel KIM-1-targeted peptide for bFGF delivery in acute kidney injury repair

doi: 10.1093/rb/rbag050

Figure Lengend Snippet: Transcriptome analysis of potential mechanism in WEV-bFGF mediating ischemic renal repair. ( A ) Volcano plots of gene differences between the WEV group and the PBS group; ( B ) KEGG analysis between the WEV group and the PBS group; ( C ) Volcano plots of gene differences between the WEV-BFGF group and the bFGF group; ( D ) KEGG analysis between the WEV-bFGF group and the bFGF group; ( E ) Heat map of differentially expressed genes related to repair and regeneration of AKI; ( F ) Quantitative PCR validation of key gene expression. All quantitative data are presented as mean ± SD, * P < 0.05, ** P < 0.01, N = 6.

Article Snippet: Subsequently, in accordance with the instructions of the Human bFGF ELISA Kit (EK0441, Boster, China), the protein extracted from kidney tissue and the content of bFGF in serum were determined.

Techniques: Real-time Polymerase Chain Reaction, Biomarker Discovery, Gene Expression

Journal: Molecular medicine reports

Article Title: Analysis of murine and human Treg subsets in inflammatory bowel disease.

doi: 10.3892/mmr.2017.6912

Figure Lengend Snippet: Figure 5. Serum inflammatory cytokines in patients with ulcerative colitis. Serum from ulcerative colitis patients and healthy donors were collected and inflammatory cytokines were determined by ELISA. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 as indicated. IL, interleukin; TSLP, thymic stromal lymphopoietin; MMP, matrix metalloproteinase.

Article Snippet: Serum cytokine levels were analyzed using the following commercially available ELISA kits according to the manufacturer's protocol: MMP-2, Total MMP-2 Quantikine ELISA kit (cat. no. MMP200; Quantikine, R&D Systems, Inc., Minneapolis, MD, USA); thymic stromal lymphopoietin, Legend MaxTM Human TSLP ELISA kit with Pre-coated Plates (cat. no. 434208; BioLegend, Inc., San Diego, CA, USA); MMP-9, Legend MaxTM Human MMP-9 ELISA kit with Pre-coated Plates (cat. no. 440707; BioLegend, Inc.); IL-17A, Legend MaxTM Human IL-17A ELISA kit with Pre-coated Plates (cat. no. 433918; BioLegend, Inc.); IL-25, Human IL-17E/IL-25 AccuSignal ELISA kit (cat. no. KOA0468; Rockland Immunochemicals, Inc., Limerick, PA, USA); and eotaxin, Human Eotaxin ELISA kit (cat. no. KOA0159; Rockland Immunochemicals, Inc.).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: ODN 2088 modulates the release of chemokines by SC astrocytes, in vitro. a Representative chemokine arrays used to detect chemokines in CM of vehicle- and ODN 2088-treated astrocytes. The chemokine arrays were independently repeated twice, showing similar results. Results from a representative experiment are shown. The dots enclosed in rectangular boxes show chemokines whose levels were decreased (> 5% difference) in the CM of ODN 2088-treated astrocytes compared to the CM of vehicle-treated astrocytes. The dots enclosed in the oval box show the chemokine whose levels were increased (> 5% difference) in the CM of ODN 2088-treated astrocytes compared to CM of vehicle-treated astrocytes. 1: CCL1; 2: CCL9/MIP-1γ; 3: CCL2/MCP-1; 4: CCL20/MIP-3α; 5: CX3CL1. b Densitometric quantification of the signal obtained in the chemokine array using the Image Lab software (Bio-Rad). c Quantification of CCL9 levels in CM obtained from ODN 2088- or vehicle-treated astrocytes [** p < 0.01, independent-sample t -test, two-tailed]. The experiment was independently repeated four times, and the mean of 4 experiments ( n = 4) is shown. d Quantification of CCL2 levels in CM obtained from ODN 2088- or vehicle-treated astrocytes [* p < 0.05, independent-sample t -test, two-tailed]. The experiment was independently repeated three times, and the mean of 3 experiments ( n = 3) is shown

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: In Vitro, Software, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: CCL1 released by ODN 2088-treated astrocytes mediates the chemotaxis of peritoneal macrophages. Quantification of F4/80 + cells that crossed to the lower surface of the membrane in response to CM derived from vehicle- or ODN 2088-treated TLR9 astrocytes in the absence or presence of CCL1 neutralizing antibody or IgG 2A isotype control [ F (5, 48) = 81.03, p < 0.0001 by one-way ANOVA, **** p < 0.0001 by Tukey’s post hoc test]. The results of three independent experiments ( n = 3) are shown. Data are presented as mean ± SEM

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: Chemotaxis Assay, Membrane, Derivative Assay, Control

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: Astrocyte-derived CCL2 and CCL9 but not CCL1 regulate macrophage polarization, in vitro. a Macrophage cultures were exposed to ODN 2088-treated astrocyte CM (ODN 2088-CM), in the absence or presence of CCL1 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [ p = 0.7228, independent-sample t -test, two-tailed]. b Macrophage cultures were exposed to vehicle-treated astrocyte CM, in the absence or presence of CCL2 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [** p < 0.01, independent-sample t -test, two-tailed]. c Macrophage cultures were exposed to vehicle-treated astrocyte CM, in the absence or presence of CCL9 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [*** p < 0.001, independent-sample t -test, two-tailed]. d Macrophage cultures were exposed to vehicle-treated astrocyte CM (Veh-CM) or ODN 2088-treated astrocyte CM (ODN 2088-CM) for 24 h, with or without (control) addition of rmCCL9 (20 pg/ml). The graph shows the quantification of the F4/80 + /Arg-1 + double-labeled cells expressed as percent of total F4/80 + cells in macrophage cultures [ F (2, 6) = 53.68, p < 0.0001 by one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 by Tukey’s post hoc test]. The experiments were independently repeated twice, yielding similar results. Results from a representative experiment are shown. Results obtained from additional biological repeats of these experiments can be found in Additional file D-G. Data are presented as mean ± SEM

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: Derivative Assay, In Vitro, Two Tailed Test, Control, Labeling

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: A scheme summarizing the effects of ODN 2088-treated astrocytes on macrophages. TLR9 antagonism increases the release of CCL1 by astrocytes, which enhances macrophage chemotaxis. In contrast, CCL2 and CCL9 release are decreased in response to ODN 2088. This reduces the negative regulatory effect of CCL2 and CCL9 on M2 macrophage polarization and fosters the M2 phenotype

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: Chemotaxis Assay

Journal: Frontiers in Immunology

Article Title: The diagnostic accuracy of CC chemokine ligand 23 for Kawasaki disease

doi: 10.3389/fimmu.2026.1758367

Figure Lengend Snippet: Intergroup comparison of CCL23 protein expression among the Kawasaki disease (KD) group, fever control (FC) group, and healthy control (HC) group.

Article Snippet: Meanwhile, the quantitative detection of CCL23 protein was conducted using the Human CCL23 ELISA Kit (Boster Biological Technology, Product No.: EK1224) in strict accordance with the kit instructions.

Techniques: Comparison, Expressing, Control

Journal: Frontiers in Immunology

Article Title: The diagnostic accuracy of CC chemokine ligand 23 for Kawasaki disease

doi: 10.3389/fimmu.2026.1758367

Figure Lengend Snippet: Diagnostic accuracy analysis of CC Chemokine Ligand 23 (CCL23) for Kawasaki disease (KD). (A) Comparison between the KD group and the fever control (FC) group; (B) Comparison between the KD group and the healthy control (HC) group; (C) Comparison between the KD group and the combined FC and HC groups.

Article Snippet: Meanwhile, the quantitative detection of CCL23 protein was conducted using the Human CCL23 ELISA Kit (Boster Biological Technology, Product No.: EK1224) in strict accordance with the kit instructions.

Techniques: Diagnostic Assay, Comparison, Control

Journal: Journal for immunotherapy of cancer

Article Title: EBV promotes TCR-T-cell therapy resistance by inducing CD163+M2 macrophage polarization and MMP9 secretion.

doi: 10.1136/jitc-2023-008375

Figure Lengend Snippet: Figure 2 EBV-infected tumors secrete CCL5 to recruit T cells and monocytes. (A, B) Representative images (left) and quantification (right) of CD8+T cells (A) or CD14+monocytes (B) recruited to the lower chamber by EBV-infected or EBV- uninfected tumor supernatants. Magnification: ×400. Mean±SD, n=3, two-tailed t-test. (C, D) Left: Representative images of EBV-uninfected or EBV-infected nasopharyngeal and gastric cancer pathology sections stained with antibodies targeting CCL5 (C) or CXCL10 (D). Magnification: ×200. Right: Measurement of CCL5 (C) and CXCL10 (D) levels in the supernatant of EBV-uninfected or EBV-infected nasopharyngeal and gastric cancer cell lines by ELISA. Mean±SD, n=4, two-tailed t-test. (E, F) Quantification of CD8+T cells (E) or CD14+ monocytes (F) recruited to the lower chamber by 200 nM CCL5, 200 nM CXCL10 or serum-free RPMI 1640. CD8+T cells and CD14+monocytes were pretreated overnight with AGS-EBV or HK1-EBV cell supernatants. Mean±SD, n=3, one-way ANOVA. (G, I) Quantification of CD8+T cells (G) and CD14+monocytes (I) recruited to the lower chamber. CD8+T cells and CD14+monocytes were pretreated overnight with AGS-EBV or HK1-EBV cell supernatant, and anti-CCR5 or CCL5+anti-CCR5 groups were simultaneously treated with 200 nM CCR5 antibody overnight to block CCR5. CD8+T cells or CD14+ monocytes were then recruited with 200 nM CCL5 or serum-free RPMI 1640. Mean±SD, n=3, one-way ANOVA. (H, J) CD8+T cells or CD14+ monocytes were treated with or without CCR5 antibody overnight to block CCR5. The above cells were recruited with EBV-infected or EBV-uninfected tumor supernatants. Quantification of CD8+T cells (H) and CD14+ monocytes (J) recruited to the lower chamber. Mean±SD, n=3, one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; EBV, Epstein-Barr virus.

Article Snippet: We used a CCL5 ELISA kit (Proteintech, KE00093), CXCL10 ELISA kit (Neobioscience, EHC157.96), CSF1 ELISA kit (Neobioscience, EHC028.96), IL10 ELISA kit (Neobioscience, EHC157.96) and MMP9 ELISA kit (Neobioscience, EHC115.96) to measure the levels of CCL5, CXCL10, CSF1, IL10 and MMP9 in the cell supernatant according to the manufacturer’s protocol.

Techniques: Infection, Two Tailed Test, Staining, Enzyme-linked Immunosorbent Assay, Blocking Assay, Virus

Journal: Journal for immunotherapy of cancer

Article Title: EBV promotes TCR-T-cell therapy resistance by inducing CD163+M2 macrophage polarization and MMP9 secretion.

doi: 10.1136/jitc-2023-008375

Figure Lengend Snippet: Figure 3 EBV-infected tumors promote CD163+M2 macrophages polarization. (A) Flow cytometry analysis of the CD163+M2 phenotype in THP-1 or CD14+ monocytes treated with EBV-infected or EBV-uninfected tumor supernatants. Mean±SD, n=4 (HK1), n=3 (AGS), two-tailed t-test. (B) Level of CSF1 in EBV-infected and EBV-uninfected tumor supernatants as measured by ELISA. Mean±SD, n=4, two-tailed t-test. (C) Flow cytometry analysis of the CD163+M2 phenotype of CD14+monocytes treated with 50 ng/mL CSF1, 80 ng/mL IL10, or both together. Mean±SD, n=3, one-way ANOVA. (D) Level of IL10 in EBV- infected and EBV-uninfected tumor supernatants as measured by ELISA. Mean±SD, n=4, two-tailed t-test. (E) Levels of IL10 in supernatants of mononuclear macrophages treated with EBV-infected and EBV-uninfected tumor supernatants for 3 days as measured by ELISA. Mean±SD, n=3, two-tailed t-test. (F) Levels of IL10 in mononuclear macrophage supernatants treated for 3 days with EBV-infected tumor supernatant, EBV-infected tumor supernatant and CSF1R inhibitor, 50 ng/mL CSF1, or no treatment as measured by ELISA. Mean±SD, n=4, two-tailed t-test. (G) Flow cytometry analysis of the CD163+M2 phenotype of CD14+monocytes treated with EBV-infected tumor supernatant (control), EBV-infected tumor supernatant and CSF1R inhibitor, EBV-infected tumor supernatant and anti-IL10R antibody, or EBV-infected tumor supernatant and CSF1R inhibitor and anti- IL10R antibody. Mean±SD, n=3, one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; EBV, Epstein-Barr virus.

Article Snippet: We used a CCL5 ELISA kit (Proteintech, KE00093), CXCL10 ELISA kit (Neobioscience, EHC157.96), CSF1 ELISA kit (Neobioscience, EHC028.96), IL10 ELISA kit (Neobioscience, EHC157.96) and MMP9 ELISA kit (Neobioscience, EHC115.96) to measure the levels of CCL5, CXCL10, CSF1, IL10 and MMP9 in the cell supernatant according to the manufacturer’s protocol.

Techniques: Infection, Flow Cytometry, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Control, Virus

Journal: Journal for immunotherapy of cancer

Article Title: EBV promotes TCR-T-cell therapy resistance by inducing CD163+M2 macrophage polarization and MMP9 secretion.

doi: 10.1136/jitc-2023-008375

Figure Lengend Snippet: Figure 6 MMP9 secreted by EBV-induced CD163+M2 macrophages suppresses TCR-T-cell function in vitro. (A) Levels of MMP9 in the supernatants of mononuclear macrophages treated with different conditions (left) as well as in EBV-infected or EBV-uninfected tumor supernatants (right) measured by ELISA. Mean±SD, n=4, two-tailed t-test. (B) Left: Representative images of EBV-positive or EBV-negative nasopharyngeal and gastric cancer tissues stained with MMP9 antibody. Magnification: ×200. Right: IHC scores of MMP9. Mean±SD, n=5 (GC and NPC+), n=4 (NPC−), two-tailed t-test. (C) Flow cytometry analysis of functional molecules in TCR-T cells after 3 days of coculture with M2 macrophages induced by CSF1+IL10, M2 macrophages and MMP9 inhibitors, untreated monocytes (control), or untreated monocytes and MMP9 inhibitors (without C666-1-A11- LMP2A). Mean±SD, n=4, one-way ANOVA. (D) Images of C666-1-A11-LMP2A cells killed by TCR-T cells at 0 hour and 16 hours which were cocultured with M2 macrophages, M2 macrophages+MMP9 inhibitor, untreated monocytes or untreated monocytes+MMP9 inhibitor for 3 days and then isolated via MACS. Magnification: ×100. (E) Statistical analysis of LDH released from C666-1-A11-LMP2A cells after 16 hours of killing by TCR-T cells with different treatment. Mean±SD, n=5, two-tailed t-test. (F) Apoptosis analysis of C666-1-A11-LMP2A cells killed by TCR-T cells for 16 hours which were cocultured with M2 macrophages, M2 macrophages+MMP9 inhibitor, untreated monocytes or untreated monocytes+MMP9 inhibitor for 3 days and then isolated via MACS Mean±SD, n=4, two-tailed t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; EBV, Epstein-Barr virus; IHC, immunohistochemistry.

Article Snippet: We used a CCL5 ELISA kit (Proteintech, KE00093), CXCL10 ELISA kit (Neobioscience, EHC157.96), CSF1 ELISA kit (Neobioscience, EHC028.96), IL10 ELISA kit (Neobioscience, EHC157.96) and MMP9 ELISA kit (Neobioscience, EHC115.96) to measure the levels of CCL5, CXCL10, CSF1, IL10 and MMP9 in the cell supernatant according to the manufacturer’s protocol.

Techniques: Cell Function Assay, In Vitro, Infection, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Staining, Flow Cytometry, Functional Assay, Control, Isolation, Virus, Immunohistochemistry

Journal: Signal Transduction and Targeted Therapy

Article Title: Intracellular CYTL1, a novel tumor suppressor, stabilizes NDUFV1 to inhibit metabolic reprogramming in breast cancer

doi: 10.1038/s41392-021-00856-1

Figure Lengend Snippet: Loss of CYTL1 switches metabolism reprogramming to glycolysis in breast cancer. a Analysis of cytl1 expression in human breast cancer issues ( n = 1085) and adjacent normal tissues ( n = 112) based on TCGA dataset. b The immunohistochemical images of breast cancer samples in different grades. Each grade shows three samples. The statistical relative intensity was evaluated by immunoreactivity intensities multiplied by the proportion of stain-positive cells, which was divided into 5 grades 0 (<5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%). c Analysis of the relevance of CYTL1 protein expression with the survival rate of breast cancer patients. d Gene set enrichment analysis (GSEA) showing glycolysis gene signatures enriched in breast cancer patients with low cytl1 expression relative to those with high cytl1 expression based on TCGA database. Breast cancer samples were divided into cytl1 -low and cytl1 -high expression groups according to the median value. NES normalized enrichment score, FDR false discovery rate. e Glucose-uptake activity in MDA-MB-231 cells transfected with HA-tagged CYTL1 expressing plasmid was detected by flow cytometry. The protein levels of CYTL1 in the transfected cells were determined using anti-CYTL1 antibody by western blot (left panel). GAPDH was used as a loading control. f Glucose-uptake activity in CYTL1 KO cells. g Glucose-uptake ability in the indicated breast cancer cell lines with different levels of intact CYTL1 expression. The protein levels of CYTL1 in these cells were determined by western blot (upper panel). Tubulin was used as a loading control. The densitometry of the immunoblots was performed with the Image J software and is presented in the histograms. h The OCR (upper panel) and ECAR (lower panel) in MDA-MB-231 cells overexpressing CYTL1 were analyzed by the Seahorse. a, e: oligomycin, b: FCCP, c: antimycin, d: glucose, f: 2-DG. i The OCR (upper panel) and ECAR (lower panel) in CYTL1 KO cells. j The OCR (upper panel) and ECAR (lower panel) in the indicated breast cancer cell lines. k , l Lactate production levels in MDA-MB-231 cells transfected with k HA-tagged CYTL1 expressing plasmids or l the indicated shRNAs were detected by a spectrophotometer and normalized to the cell number. m Lactate production levels in the indicated breast cancer cell lines. The data are shown as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Human CYTL1 ELISA Kit was purchased from CUSABIO Life Sciences (College Park, MD).

Techniques: Expressing, Immunohistochemical staining, Staining, Activity Assay, Transfection, Plasmid Preparation, Flow Cytometry, Western Blot, Control, Software, Spectrophotometry

Journal: Signal Transduction and Targeted Therapy

Article Title: Intracellular CYTL1, a novel tumor suppressor, stabilizes NDUFV1 to inhibit metabolic reprogramming in breast cancer

doi: 10.1038/s41392-021-00856-1

Figure Lengend Snippet: Intracellular CYTL1 inhibits glycolysis in breast cancer. a Glucose-uptake activity and b lactate production levels in MDA-MB-231 cells after treatment with rhCYTL1 at the indicated concentrations for 48 h. ns no significant. c The subcellular location of CYTL1 was analyzed by immunofluorescence in MDA-MB-231 cells. Representative data are shown from three independent experiments. Scale bar, 5 μm. d The amount of CYTL1 was determined by western blot in MDA-MB-231 cells after treatment with monensin for the indicated period. Tubulin was used as a loading control. e The OCR and f lactate production levels in MDA-MB-231 cells after treatment with 1 μM monensin for 24 h. g The supernatants and cell lysates from MDA-MB-231 cells transfected with HA-tagged CYTL1 or ΔCYTL1 expressing plasmids were determined by western blot using an anti-HA antibody. Right panel: schematic representation of the full-length CYTL1 and ΔCYTL1 plasmids. h Glucose-uptake activity and i lactate production levels in MDA-MB-231 cells overexpressing ΔCYTL1. The data are shown as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Human CYTL1 ELISA Kit was purchased from CUSABIO Life Sciences (College Park, MD).

Techniques: Activity Assay, Immunofluorescence, Western Blot, Control, Transfection, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Intracellular CYTL1, a novel tumor suppressor, stabilizes NDUFV1 to inhibit metabolic reprogramming in breast cancer

doi: 10.1038/s41392-021-00856-1

Figure Lengend Snippet: Intracellular CYTL1 prevents tumor growth and metastases. a Cell numbers were counted using a Trypan exclusion assay after MDA-MB-231 cells expressing ΔCYTL1 were cultured for the indicated period. b Colony formation assay was performed after MDA-MB-231 cells expressing ΔCYTL1 were cultured for 15 days. c Cell migration of MDA-MB-231 cells expressing ΔCYTL1 was determined using transwell assay. d – h Female nude mice were subjected to orthotopic injection with MDA-MB-231 cells stably expressing ΔCYTL1. Some mice were sacrificed 28 days later, after which tumors, lungs, and sera were collected, followed by photography and measurement. Some mice were monitored until they died. d Tumor growth curves ( n = 6). Upper panel: representative images of the tumors at the end of the experiments. e Tumor weight. f Survival curve of mice ( n = 6). g Serum lactate levels. h Representative H&E-stained lung sections. Circles indicate metastatic foci in the lungs. Scale bar, 500 μm. Lower panel: the proportion of mice with lung metastases in each group. i – l Female C57BL/6 mice were subjected to orthotopic injection with E0771 cells stably expressing ΔCYTL1. The mice were sacrificed 36 days later, after which tumors, lungs, and sera were collected. i Tumor growth curves ( n = 6). Upper panel: representative images of the tumors at the end of the experiments. j Tumor weight. k Serum lactate levels. l Representative H&E-stained lung sections. Circles indicate metastatic foci in the lungs. Scale bar, 500 μm. Lower panel: the proportion of mice with lung metastases in each group. The data are shown as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Human CYTL1 ELISA Kit was purchased from CUSABIO Life Sciences (College Park, MD).

Techniques: Exclusion Assay, Expressing, Cell Culture, Colony Assay, Migration, Transwell Assay, Injection, Stable Transfection, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: Intracellular CYTL1, a novel tumor suppressor, stabilizes NDUFV1 to inhibit metabolic reprogramming in breast cancer

doi: 10.1038/s41392-021-00856-1

Figure Lengend Snippet: Intracellular CYTL1 interacts with NDUFV1 and positively regulates its protein expression. a ATP and ADP levels and b NAD + and NADH levels in the cell lysates from MDA-MB-231 cells stably expressing CYTL1 or ΔCYTL1 were determined using kit assays. c Co-IP analysis of the interaction between exogenous HA-CYTL1 or HA-ΔCYTL1 and exogenous Flag-NDUFV1 in transfected HEK293T cells. d Co-IP analysis of the interaction between endogenous NDUFV1 and exogenous HA-CYTL1 or HA-ΔCYTL1 in transfected HEK293T cells. IgG served as the negative control. Tubulin was used as a loading control. e Colocalization of HA-CYTL1 and endogenous NDUFV1 was detected by Immunofluorescence in transfected HEK293T cells. Scale bar, 5 μm. f , g NDUFV1 protein expression was determined by western blot in MDA-MB-231 cells transfected with f HA-CYTL1 or HA-ΔCYTL1 expressing plasmids or g the indicated shRNAs. Tubulin was used as a loading control. h Lactate production levels in CYTL1 or ΔCYTL1 overexpressing MDA-MB-231 cells cotransfected with the indicated shRNAs. i Glucose-uptake activity and j the OCR in MDA-MB-231 cells overexpressing NDUFV1. The data are shown as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Human CYTL1 ELISA Kit was purchased from CUSABIO Life Sciences (College Park, MD).

Techniques: Expressing, Stable Transfection, Co-Immunoprecipitation Assay, Transfection, Negative Control, Control, Immunofluorescence, Western Blot, Activity Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Intracellular CYTL1, a novel tumor suppressor, stabilizes NDUFV1 to inhibit metabolic reprogramming in breast cancer

doi: 10.1038/s41392-021-00856-1

Figure Lengend Snippet: Intracellular CYTL1 interrupts MDM2-mediated degradation of NDUFV1 by competitive binding. a The degradation of NDUFV1 over time in the presence of cycloheximide (CHX) was monitored by western blot in MDA-MB-231 cells transfected with HA-CYTL1 or HA-ΔCYTL1 expressing plasmids. b The NDUFV1 protein level in MDA-MB-231 cells before and after treatment of 20 μM MG132 for 6 h. c Co-IP analysis of the interaction between Flag-NDUFV1 and HA-Ub in transfected HEK293T cells. d Co-IP analysis of the interaction between NDUFV1 and HA-Ub in HEK293T cells cotransfected with Myc-NC or Myc-CYTL1 after treatment of 20 μM MG132 for 6 h. e , f NDUFV1 protein expression was determined by western blot in MDA-MB-231 cells transfected with e MDM2 expressing plasmid or f siRNA specific for MDM2. Tubulin was used as a loading control. g Co-IP analysis of the interaction between MDM2 and Flag-NDUFV1 in transfected HEK293T cells. h Co-IP analysis of the interaction between MDM2 and Flag-NDUFV1 in transfected HEK293T cells cotransfected with HA-NC, HA-CYTL1, or HA-ΔCYTL1. i Schematic representation of NDUFV1 mutants that have truncated sequences. j Co-IP analysis of the interaction between HA-CYTL1 and Flag-NDUFV1 or its three mutants in transfected HEK293T cells. Stars indicate non-specific bands. k Co-IP analysis of the interaction between HA-CYTL1 and Flag-NDUFV1 or its two mutants with deletion of N-terminal sequences. l Co-IP analysis of the interaction between MDM2 and Flag-NDUFV1 or its two mutants. m Cytoplasmic and mitochondrial protein were extracted from HEK293T cells transfected with HA-NC or HA-CYTL1. Co-IP analysis was performed for the interaction between endogenous NDUFV1 and HA-CYTL1. The data are shown as the mean ± SD of three independent experiments. *** P < 0.001

Article Snippet: Human CYTL1 ELISA Kit was purchased from CUSABIO Life Sciences (College Park, MD).

Techniques: Binding Assay, Western Blot, Transfection, Expressing, Co-Immunoprecipitation Assay, Plasmid Preparation, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Intracellular CYTL1, a novel tumor suppressor, stabilizes NDUFV1 to inhibit metabolic reprogramming in breast cancer

doi: 10.1038/s41392-021-00856-1

Figure Lengend Snippet: NDUFV1 suppresses the Y10 phosphorylation of LDHA by Src. a LDHA enzyme activity in MDA-MB-231 cells overexpressing CYTL1 was detected using kit assay. b LDHA enzyme activity in CYTL1 KO cells. c LDHA enzyme activity in MDA-MB-231 cells overexpressing NDUFV1. d , e LDHA Y10 phosphorylation level was determined by western blot in the indicated cells transfected with d Flag-NDUFV1 expressing plasmids or e the shRNAs against NDUFV1. GAPDH was used as a loading control. f MDA-MB-231 orthotopic breast cancer models were established as described in Fig. . The indicated proteins in tumor tissues were determined by western blot. g Co-IP analysis of the interaction between Flag-tagged NDUFV1 and endogenous Src in transfected HEK293T cells. h The levels of phosphorylated Src and total Src were determined by western blot in MDA-MB-231 cells overexpressing NDUFV1. i Co-IP analysis of the interaction between Src and LDHA in HEK293T cells transfected with Flag-NDUFV1 expressing plasmids. j A schematic model for the role of intracellular CYTL1 in regulating metabolic reprogramming in breast cancer. The data are shown as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Human CYTL1 ELISA Kit was purchased from CUSABIO Life Sciences (College Park, MD).

Techniques: Phospho-proteomics, Activity Assay, Western Blot, Transfection, Expressing, Control, Co-Immunoprecipitation Assay

Journal: Free radical biology & medicine

Article Title: Oxidative stress-induced decreased expression of FABP5 leads to mitochondrial damage and survival disorder of decidual stromal cells in women with recurrent spontaneous abortion.

doi: 10.1016/j.freeradbiomed.2025.06.003

Figure Lengend Snippet: Fig. 7. FABP5 downregulation in DSCs repressed CXCL11/CXCR3 signaling between DSCs and HTR-8/Svneo cells. (A) KEGG pathway analysis of DEGs identified by RNA-seq in FABP5-knockdown primary DSC. (B) Gene Set Enrichment Analysis (GSEA) of chemokine signaling pathways in FABP5-silenced DSCs. (C) FPKM values of CXCL11 expression in control and FABP5-knockdown groups. (D) RT-qPCR, (E, F) Western blot, and (G) ELISA quantification of CXCL11 expression in DSCs transfected with FABP5-targeting or NC siRNAs. (H) RT-qPCR and (I, J) Western blot analyses of CXCR3 expression in HTR-8/Svneo cells treated with CS from FABP5-deficient or control DSCs. Statistical significance: *P < 0.05, **P < 0.01.

Article Snippet: The concentration of CXCL11 in the CS was measured using a human CXCL11 ELISA kit (Boster, Beijing, China; CAT# EK0737) following the manufacturer’s instructions.

Techniques: RNA Sequencing, Knockdown, Protein-Protein interactions, Expressing, Control, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection