Journal: JCI Insight

Article Title: CSF1/CSF1R signaling mediates malignant pleural effusion formation

doi: 10.1172/jci.insight.155300

Figure Lengend Snippet: ( A and B ) CAFs in tumors of vehicle- and CSF1Ri-treated animals were visualized upon FAPA staining. ( A ) Representative pictures. Scale bar: 50 μm. ( B ) Results of image analysis. Data presented as mean ± SEM. LLC: n = 4 for both groups. MC38: n = 5 for both groups. * P < 0.05 compared with vehicle by 2-tailed Student’s t test. HPF, high-power field. ( C ) Activated CAFs isolated from LLC or MC38 tumors were treated with vehicle or 6700 nM CSF1Ri overnight and subsequently loaded with 7 × 10 4 tumor cells/well; 8 hours later 2 × 10 5 M1 macrophages/well were added to the tumor cell–CAF cocultures; 24 hours later, tumor cells were counted. ( D and E ) CD45 + CD11b + F4/80 + macrophages were analyzed for IL-10 ( D ) and IL-12 ( E ) expression. Data presented as mean ± SEM, 2 independent experiments. LLC: vehicle-treated CAFs n = 5, CSF1Ri-treated CAFs n = 5. MC38: vehicle-treated CAFs n = 7, CSF1Ri-treated CAFs n = 7. * P < 0.05 compared with vehicle-treated CAFs by 2-tailed Student’s t test. ( F and G ) Isolated CAFs from LLC and MC38 tumors (treated with vehicle or CSF1Ri overnight) were cocultured with macrophages for 48 hours. Macrophages were isolated and mRNA levels of Vegfa ( F ) and Il6 ( G ) were quantified by real-time PCR. Data presented as mean ± SEM. n = 7 for both groups, * P < 0.05 compared with indicated group by 2-tailed Student’s t test. ( H ) Isolated CAFs from LLC or MC38 tumors were treated with vehicle, CSF1Ri, CSF1, or IL-34 or pretreated with CSF1Ri for 2 hours and then CSF1 was added; 24 hours later, MIP2 levels were quantified in supernatants. Results were normalized to total protein. Data presented as mean ± SEM. Vehicle n = 6, CSF1Ri n = 6, CSF1 n = 5, CSF1Ri+CSF1 n = 4, IL-34 n = 5 (3 independent experiments). * P < 0.05 compared with indicated group by 1-way ANOVA (with Tukey’s post hoc test).

Article Snippet: CAFs were isolated from LLC or MC38 tumors by FAPA antibody–loaded (CSB-PA008191, Cusabio Technology) magnetic beads (MojoSort Streptavidin Nanobeads, BioLegend) as previously reported ( ).

Techniques: Staining, Isolation, Expressing, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: CXCL3/TGF-β-mediated crosstalk between CAFs and tumor cells augments RCC progression and sunitinib resistance

doi: 10.1016/j.isci.2024.110224

Figure Lengend Snippet:

Article Snippet: Rabbit Anti-FAP Rabbit Polyclonal Antibody , Solarbio life science , Cat# K004451P; RRID:AB_2313773.

Techniques: Recombinant, Cell Culture, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, RNA Extraction, Sequencing, Over Expression, Plasmid Preparation, Software

Journal:

Article Title: FAP-1 Association with Fas (Apo-1) Inhibits Fas Expression on the Cell Surface

doi: 10.1128/MCB.23.10.3623-3635.2003

Figure Lengend Snippet: Fas and FAP-1 expression in human melanoma cell lines. (A) Surface and total (after cell permeabilization) Fas levels were determined by staining with PE-conjugated anti-human Fas MAbs and flow cytometry of melanoma cells. (B) Immunostaining of endogenous Fas in WM9 and OM431 melanoma cells. Anti-Fas MAb and fluorescein isothiocyanate-labeled secondary Abs were used. DAPI was used for nuclear staining. The merged images were obtained by confocal microscopy. (C) RT-PCR analysis of FAP-1 mRNA levels in human melanomas. GAPDH mRNA levels were used as an internal control. (D) Western blot analysis (W) of FAP-1 and Fas levels in melanomas. The positions of FAP-1 and of a nonspecific form (ns) recognized by these Abs are indicated by arrowheads. The β-actin level was used as a loading control. Fas and FAP-1 proteins prepared from the indicated melanoma cell lines using RIPA buffer were subjected to coimmunoprecipitation (IP) with Abs to FAP-1 followed by immunoblot analysis with Abs to Fas and FAP-1. Apparent molecular masses (kilodaltons) are in parentheses. (E) Western blot analyses of 1% Triton X-100-soluble and -insoluble fractions from FEMX and OM431 cells were performed using Abs against Fas, FAP1, vinculin, RIP-1, and Src.

Article Snippet: The primary Abs used were rabbit anti-FAP-1 or monoclonal anti-Fas (Pharmingen), anti-β-actin (Sigma), and anti-c-Src (UBI) (1:1,000 to 1:5,000).

Techniques: Expressing, Staining, Flow Cytometry, Immunostaining, Labeling, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal:

Article Title: FAP-1 Association with Fas (Apo-1) Inhibits Fas Expression on the Cell Surface

doi: 10.1128/MCB.23.10.3623-3635.2003

Figure Lengend Snippet: FAP-1 expression attenuates Fas-GFP cell surface expression. (A) Western blot analysis of FAP-1 levels in 1% Triton X-100-soluble and -insoluble fractions in control (empty vector) or FAP-1-tranfected FEMX cells. ns, a nonspecific form recognized by the Abs. (B) Western blot analysis (W) of FAP-1 levels (RIPA buffer) in control (empty vector) or FAP-1-transfected FEMX cells. Both cell lines were cotransfected with Fas-GFP. IP, immunoprecipitation. (C) Surface Fas levels were determined in transfected FEMX cells by staining them with anti-N-terminal Fas-PE MAb and by FACS analysis. The MFIs of Fas and the percentages of double-positive Fas+ GFP+ cells are indicated. (D) Confocal images of FEMX cells transfected with Fas-GFP plus empty vector or Fas-GFP plus FAP-1 expression construct. γ-Adaptin was used as a Golgi marker.

Article Snippet: The primary Abs used were rabbit anti-FAP-1 or monoclonal anti-Fas (Pharmingen), anti-β-actin (Sigma), and anti-c-Src (UBI) (1:1,000 to 1:5,000).

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection, Immunoprecipitation, Staining, Construct, Marker

Journal:

Article Title: FAP-1 Association with Fas (Apo-1) Inhibits Fas Expression on the Cell Surface

doi: 10.1128/MCB.23.10.3623-3635.2003

Figure Lengend Snippet: Mutated forms of FAP-1 increase cell surface expression of Fas. (A) Expression levels of mutated forms of transfected FAP-1 in OM431 cells were determined by Western blot analysis (W). IP, immunoprecipitation. Apparent molecular masses (kilodaltons) are in parentheses. (B) FACS analysis of Fas-GFP surface expression was performed in OM431 cells after cotransfection with vector, FAP-1, or mutated forms of FAP-1 using anti-N-terminal Fas-PE MAbs and C-terminally tagged GFP. The MFIs and the percentages of double-positive cells (or numbers of double-positive cells of a total of 40,000 cells analyzed) are indicated in the upper right quadrants. (C) Effect of FAP-1 or FAP-1ΔPGZ3 on endogenous Fas expression in OM431 cells. The indicated constructs were cotransfected together with GFP, and 24 h later, FACS analysis allowed the analysis of endogenous Fas levels in GFP-positive cells. The ratios of Fas+ GFP+ cells to the total number of GFP+ cells are indicated.

Article Snippet: The primary Abs used were rabbit anti-FAP-1 or monoclonal anti-Fas (Pharmingen), anti-β-actin (Sigma), and anti-c-Src (UBI) (1:1,000 to 1:5,000).

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation, Cotransfection, Plasmid Preparation, Construct

Journal:

Article Title: FAP-1 Association with Fas (Apo-1) Inhibits Fas Expression on the Cell Surface

doi: 10.1128/MCB.23.10.3623-3635.2003

Figure Lengend Snippet: Mutated forms of FAP-1 and RNAi of FAP-1 increase cell surface expression of Fas. (A) FACS analysis of surface and total Fas levels in TIG3 cells. (B) Western blot analysis (W) of FAS-GFP and FAP-1 levels in TIG3 cells. IP, immunoprecipitation; ns, a nonspecific form recognized by the Abs. (C) FACS analysis of Fas-GFP surface expression was performed in TIG3 cells after cotransfection with vector, FAP-1, or dominant-negative forms of FAP-1 using anti-N-terminal Fas-PE MAbs and C-terminally tagged GFP. (D) Western blot analysis of FAP-1 levels in TIG3 cells transfected with pRS, FAP-1 RNAi, or control RNAi 60 h after transfection. (E) Immunohistochemistry using confocal microscopy follows changes in surface expression of endogenous Fas in TIG3 cells that were transfected with FAP-1 RNAi or control RNAi. (F) Changes in endogenous levels of surface Fas were determined in TIG3 cells after cotransfection of red fluorescent protein (RFP) expression vector together with either pSR or FAP-1 RNAi and control RNAi. FACS analysis was performed for the determination of the percentage of Fas-positive cells and Fas MFI in the population of GFP-positive cells. The error bars indicate standard deviations.

Article Snippet: The primary Abs used were rabbit anti-FAP-1 or monoclonal anti-Fas (Pharmingen), anti-β-actin (Sigma), and anti-c-Src (UBI) (1:1,000 to 1:5,000).

Techniques: Expressing, Western Blot, Immunoprecipitation, Cotransfection, Plasmid Preparation, Dominant Negative Mutation, Transfection, Immunohistochemistry, Confocal Microscopy

Journal:

Article Title: FAP-1 Association with Fas (Apo-1) Inhibits Fas Expression on the Cell Surface

doi: 10.1128/MCB.23.10.3623-3635.2003

Figure Lengend Snippet: Dominant-negative forms of FAP-1 further increase cell surface expression of Fas in LU1205 melanoma cells. (A) FACS analysis of Fas-GFP expression in cells expressing wt and mutant forms of FAP-1 was performed as indicated in Materials and Methods. (B) Corresponding confocal images of merged GFP-Fas and Golgi marker (γ-adaptin) are shown for the experiment depicted in panel A. (C) Western blotting of FAP-1 levels in LU1205 cells that stably express control or FAP-1ΔPDZ3 constructs. ns, a nonspecific form recognized by the Abs. (D) FACS analysis of Fas levels in LU1205 cells that stably express pcDNA3 (neo) or FAP-1ΔPDZ3. (E) Apoptosis analysis of control or FAP-1ΔPDZ3-transfected LU1205 cells after treatment with FasL (50 ng/ml) plus CHX (10 μg/ml). The percentages of apoptotic cells are indicated.

Article Snippet: The primary Abs used were rabbit anti-FAP-1 or monoclonal anti-Fas (Pharmingen), anti-β-actin (Sigma), and anti-c-Src (UBI) (1:1,000 to 1:5,000).

Techniques: Dominant Negative Mutation, Expressing, Mutagenesis, Marker, Western Blot, Stable Transfection, Construct, Transfection

Journal:

Article Title: FAP-1 Association with Fas (Apo-1) Inhibits Fas Expression on the Cell Surface

doi: 10.1128/MCB.23.10.3623-3635.2003

Figure Lengend Snippet: Mutation within C-terminal Fas abolishes FAP-1 association and inhibition of Fas trafficking. (A) Effect of N- and/or C-terminal mutations of Fas on association of Fas-GFP with FAP-1. The corresponding constructs were expressed in OM431 cells following their coimmunoprecipitation (IP) and Western analysis (W) with the indicated Abs. The positions of Fas-GFP and the imunoglobulin heavy chain (H, Ig) are indicated. (B) Immunohistochemical analysis of Fas-GFP expression in OM431 cells that were transfected with the wt or mutant forms of Fas-GFP.

Article Snippet: The primary Abs used were rabbit anti-FAP-1 or monoclonal anti-Fas (Pharmingen), anti-β-actin (Sigma), and anti-c-Src (UBI) (1:1,000 to 1:5,000).

Techniques: Mutagenesis, Inhibition, Construct, Western Blot, Immunohistochemical staining, Expressing, Transfection

Journal: Cancers

Article Title: NAB 2-Expressing Cancer-Associated Fibroblast Promotes HNSCC Progression

doi: 10.3390/cancers11030388

Figure Lengend Snippet: CAF marker and matrix metalloproteinase (MMP) expression in primary fibroblasts from HNSCC patient tissue. ( A ) CAFs and paired non-tumor fibroblasts (NTFs) were isolated from three HNSCC patient tissue (P1, P2, P3) specimens by short-term primary culture. α-SMA and FAP mRNA and protein levels were evaluated by real-time quantitative polymerase chain reaction (qPCR) and Western blotting, respectively. ( B ) MMP mRNA and protein expression was analyzed under the same conditions. Results represent the mean ± standard deviation of two or three experiments.

Article Snippet: Rabbit anti-fibroblast activation protein (FAP) antibody was from GeneTex (GeneTex, Inc., Irvine, CA, USA).

Techniques: Marker, Expressing, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation