Journal: PLoS ONE

Article Title: Human FGF-21 Is a Substrate of Fibroblast Activation Protein

doi: 10.1371/journal.pone.0151269

Figure Lengend Snippet: (A) Human FGF-21 is cleaved by FAP. Recombinant human FGF-21 was digested by recombinant human FAP and visualized by Coomassie staining of SDS-Page gel. (B) Time course of FGF-21 digestion by FAP quantified by LC/MS extracted ion integration of peaks corresponding to intact (1–181) and cleaved (1–171) forms of FGF-21 (n = 3 per time point per group). Values are mean ± SEM with one phase decay curve fit superimposed. (C) FAP cleavage of FGF-21 is prevented by ARI-3099. ARI-3099 was pre-incubated with recombinant FAP for 30 minutes prior to addition of FGF-21. Reaction products were visualized by Coomassie staining of SDS-Page gel. (D) Recombinant PREP does not cleave FGF-21. Recombinant human PREP was added to recombinant FGF-21 and visualized by Coomassie staining of SDS-Page gel.

Article Snippet: Reactions were carried out at a final concentration of 20 μM FGF-21, 200 nM recombinant human FAP (R&D systems) or PREP (R&D systems) and 16 μM ARI-3099.

Techniques: Recombinant, Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: PLoS ONE

Article Title: Human FGF-21 Is a Substrate of Fibroblast Activation Protein

doi: 10.1371/journal.pone.0151269

Figure Lengend Snippet: (A) FAP cleaves human FGF-21 in mouse, monkey and human plasma. Recombinant FGF-21 was added to plasma to a final concentration of 1 μM in the presence or absence of 16 μM ARI-3099 followed by assessment of intact FGF-21 by sandwich ELISA (n = 3 per group). Values are mean ± SEM. *P < .05 ***P < .001 by ANOVA . (B) FAP activity of mouse, monkey and human plasma as assessed by the FAP-specific fluorescent substrate ARI-3144.

Article Snippet: Reactions were carried out at a final concentration of 20 μM FGF-21, 200 nM recombinant human FAP (R&D systems) or PREP (R&D systems) and 16 μM ARI-3099.

Techniques: Clinical Proteomics, Recombinant, Concentration Assay, Sandwich ELISA, Activity Assay

Journal: Genes & Diseases

Article Title: Cancer-associated fibroblasts derived fibronectin extra domain A promotes sorafenib resistance in hepatocellular carcinoma cells by activating SHMT1

doi: 10.1016/j.gendis.2024.101330

Figure Lengend Snippet: CAFs promote sorafenib resistance by activating NF-κB in HCC cells. (A) Immunofluorescence analysis was performed to assess the expression of FAP and α-SMA on primary CAFs. Scale bars, 50 μm. (B) Co-culture with CAFs significantly reduced apoptosis of HepG2 and Huh7 upon sorafenib treatment. (blue bar: sorafenib-treated tumor cells cultured alone; red bar: sorafenib-treated tumor cells co-cultured with CAFs). Student's t -test of variance, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (C) Enrichment score of the “I-κB kinase/NF-κB signaling” in sorafenib-resistant group versus sorafenib-sensitive group after sorafenib treatment, analyzed by Gene Set Enrichment Analysis based on the RNA-seq data (after performing log 2 transformation, normalization, and mean value calculation) obtained from the GEO database (GSE182593). (D) Western blot analyses of the protein level of P-p65 in the indicated HCC cells with different treatments. Results are representative of three experiments. (E) Inhibition of the NF-κB signaling pathway in HCC cells induced apoptosis significantly upon sorafenib treatment (blue bar: sorafenib-treated group; red bar: sorafenib and BAY11-7082-treated (100 μM) group). Student's t -test of variance, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. CAFs, cancer-associated fibroblasts; NF-κB, nuclear factor kappa B; HCC, hepatocellular carcinoma; FAP, fibroblast activation protein; α-SMA, alpha-smooth muscle actin.

Article Snippet: The cells on slides were fixed with 4% paraformaldehyde for 10 min and permeabilized in phosphate buffer saline for 20 min. Then, cells were blocked with goat serum at room temperature for 60 min and incubated with primary antibodies against FAP (fibroblast activation protein; R&D system, #FAB3715A, RRID: AB_2884010) (1:200) and α-SMA (alpha-smooth muscle actin; R&D system, #MAB1420, RRID: AB_262054) (1:200) for 2 h. Then, the cells on slides were reheated and incubated with the corresponding secondary antibody at 37 °C in the dark for 2 h. Nuclei were counter-stained with DAPI.

Techniques: Immunofluorescence, Expressing, Co-Culture Assay, Cell Culture, RNA Sequencing, Transformation Assay, Western Blot, Inhibition, Activation Assay

Journal: Cell death discovery

Article Title: Exosomes derived from ccRCC cells confers fibroblasts activation to foster tumor progression through Warburg effect by downregulating PANK3.

doi: 10.1038/s41420-025-02434-8

Figure Lengend Snippet: Fig. 1 Advanced ccRCC tissues are enriched with CAFs. A UMAP plot of CAFs derived from ccRCC tissues at different stages of progression, colored by tissue origin. B IHC staining detection of FAP on ccRCC tissues. Scale bar, 50 μm. C Immunofluorescence detection of α-SMA and FAP on ccRCC tissues. Scale bar, 100 μm. D Western blotting assay of α-SMA and FAP in primary isolated NFs and CAFs. E qRT-PCR analysis of pro-inflammatory genes expression of NFs and CAFs. Each experiment was performed three times independently and results are presented as mean ± s.d. Student’s t-test was used to analyze the data (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Primary antibodies targeting α-SMA (1:2000, Abcam, #ab7817, USA) and FAP (1:1000, Cell Signaling Technology, #66562s, USA) were used.

Techniques: Derivative Assay, Immunohistochemistry, Western Blot, Isolation, Quantitative RT-PCR, Expressing

Journal: Cell death discovery

Article Title: Exosomes derived from ccRCC cells confers fibroblasts activation to foster tumor progression through Warburg effect by downregulating PANK3.

doi: 10.1038/s41420-025-02434-8

Figure Lengend Snippet: Fig. 4 The down-regulation of PANK3 induces fibroblast activation. A The process of synthesizing Coenzyme A and key enzymes. B Differential expression and prognostic analysis of PANK3 in the TCGA-KIRC database. C Multivariate COX regression analysis of PANK3 in the TCGA-KIRC database. D qRT-PCR and immunoblotting assays of PNAK3 in NF and CAF3. E qRT-PCR and immunoblotting assays of PANK3 in NF and CAF3 transfected with si PANK3 or plasmid vector containing PANK3. F Analysis of ECAR and OCR of NFs transfected with si PANK3 or si NC. G Comparison of glucose uptake, GLUT1 expression, total ROS level of NFs transfected with si PANK3 or si NC. H Comparison of lactate production of NFs transfected with si PANK3 or si NC. I Immunoblotting assays of α-SMA, FAP, and MCTs in NFs transfected with si PANK3 or si NC. J qRT-PCR analysis of pro-inflammatory genes expression of NFs transfected with si PANK3 or si NC. Each experiment was performed three times independently and results are presented as mean ± s.d. Student’s t-test was used to analyze the data (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Primary antibodies targeting α-SMA (1:2000, Abcam, #ab7817, USA) and FAP (1:1000, Cell Signaling Technology, #66562s, USA) were used.

Techniques: Activation Assay, Quantitative Proteomics, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Comparison, Expressing

Journal: Biomedicines

Article Title: Reversal of Myofibroblast Apoptosis Resistance and Collagen Deposition by Phaseoloidin-Induced Autophagy Attenuates Pulmonary Fibrosis

doi: 10.3390/biomedicines13112679

Figure Lengend Snippet: PTPN13 mediated by phaseoloidin links autophagy and apoptosis. ( A ) Western blot analysis of PTPN13 levels in FasL and different dose phaseoloidin co-treated myofibroblasts ( left ) and quantification of the PTPN13 band intensity ( right ). ( B ) Western blotting reflects the CO-IP assay of PTPN13 binding to p62 under co-treatment with FasL and phaseoloidin or CQ. ( C ) Western blot analysis of PTPN13 levels in FasL and 100 μM phaseoloidin co-treated primary mouse myofibroblasts in the presence or absence of CQ ( top ) and quantification of the PTPN13 band intensity ( bottom ). ( D ) Flow cytometry was used to detect the percentage of apoptosis in the above experimental groups. ( E ) Flow cytometry was used to detect the percentage of apoptosis in groups with or without vector-PTPN13. Data in this figure represent the means ± S.D. (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: A total of 10% of the proteins electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were transferred to PVDF membranes (Bio-Rad) and 5% ( w / v ) skim milk with TBS buffer containing 0.1% ( v / v ) Tween 20 (TBST) TBS buffer for blocking. β-ACTIN monoclonal antibody (1:5000, Proteintech, Wuhan, China), α-SMA monoclonal antibody (1:1000, CST, USA), Caspase3 polyclonal antibody (1:1000, Proteintech, China), collagen I polyclonal antibody (1:1000, Millipore, USA), Bcl-2 polyclonal antibody (1:1000, Proteintech, China), BAX polyclonal antibody (1:1000, Proteintech, China), PTPN13 polyclonal antibody (1:1000, RD, Minneapolis, MN, USA), p70S6K polyclonal antibody (1:1000, CST, USA), elf-4B polyclonal antibody (1:500, ABclonal, Wuhan, China), mTOR monoclonal antibody (1:1000, CST, USA), Pho-mTOR monoclonal antibody (1:1000, CST, USA), AMPK monoclonal antibody (1:1000, CST, USA) and Pho-AMPK monoclonal antibody (1:1000, CST, USA) were incubated overnight at 4 °C.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Flow Cytometry, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Colorectal cancer cell intrinsic fibroblast activation protein alpha binds to Enolase1 and activates NF-κB pathway to promote metastasis

doi: 10.1038/s41419-021-03823-4

Figure Lengend Snippet: A Immunofluorescence analysis of FAP location in HCT116-FAP and HCT8-FAP cells. Error bar = 30 μm. B ELISA assay detects the concentration of FAP in media of HCT116-FAP, HCT8-FAP, HCT116-Ctrl, and HCT8-Ctrl cells. Ctrl: control; FAP: FAP overexpression. C Migration assays of HCT116 and HCT8 cells treated with control medium and FAP-overexpressed medium. Error bar = 100 μm. * p < 0.05, *** p < 0.001. The CM was added into the upper chamber. CM: conditioned medium. D Invasion assays of HCT116 and HCT8 cells treated with control medium and FAP-overexpressed medium. Error bar = 100 μm. * p < 0.05, *** p < 0.001. The CM was added into the upper chamber. E Migration assays of HCT116 and HCT8 cells treated with different concentration of recombinant FAP. Error bar = 100 μm. ** p < 0.01, *** p < 0.001. F Invasion assays of HCT116 and HCT8 cells treated with different concentration of recombinant FAP. Error bar = 100 μm. ** p < 0.01, *** p < 0.001.

Article Snippet: FAP concentration was detected using FAP DuoSet ELISA kit (R&D Systems, DY3715) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Enzyme-linked Immunosorbent Assay, Concentration Assay, Control, Over Expression, Migration, Recombinant

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Breast cancer patient-derived microtumors resemble tumor heterogeneity and enable protein-based stratification and functional validation of individualized drug treatment

doi: 10.1186/s13046-023-02782-2

Figure Lengend Snippet: Antibodies for IHC staining

Article Snippet: rabbit anti-human FAPalpha , BioRad , AHP1322 , Rb Linker , 1:50.

Techniques:

Journal: Current Issues in Molecular Biology

Article Title: Oncological Safety of High Hydrostatic Pressure Treatment: Effects on Cancer-Associated Fibroblast-like Transdifferentiation of Adipose Stromal Cells

doi: 10.3390/cimb48010091

Figure Lengend Snippet: Protein expression analysis of cancer-associated fibroblast (CAF) markers in stimulated human adipose stromal cells (hASCs). HASCs were incubated for 96 h with tumor-conditioned medium (TCM), 0 MPa-CM, or 300 MPa-CM derived from HNSCC16 or UTSCC14 cells, TGF-β1 (10 ng/mL), or serum-free control medium, respectively. ( A ) α-SMA protein expression was determined by Western blot analysis. Representative Western blot membrane with protein bands of α-SMA following incubation with primary and secondary antibody is shown. Precision Plus Protein™ All Blue prestained standard served as molecular weight marker. Densitometric quantification of α-SMA protein expression was evaluated and total protein normalization was performed using Bio-Rad Stain-Free™ technology. Data represents n = 4 independent donors (mean ± SD). Results are presented as relative protein expression normalized to the serum-free control (set to 1; illustrated as dashed lines). ( B ) FAP levels were determined by ELISA in hASCs from six independent donors after 96 h of stimulation with the respective conditioned media. Results are presented as relative protein expression normalized to the serum-free control (set to 1; illustrated as dashed lines). Statistical significance was assessed using predefined post hoc comparisons following one-way ANOVA or Kruskal–Wallis testing. Differences were evaluated relative to the control group or between specific treatment conditions as indicated in the figures. Statistically significant differences are indicated by asterisks (* p < 0.05, ** p < 0.01).

Article Snippet: Determination of FAP concentration was performed on cell lysates using the DuoSet Human FAP enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Incubation, Derivative Assay, Control, Western Blot, Membrane, Molecular Weight, Marker, Staining, Enzyme-linked Immunosorbent Assay