Journal: Clinical and Translational Medicine

Article Title: Neuro‐associated SEMA3C + tumour cells promote gastric cancer peritoneal metastasis via EBF2–FGF7–FGFR2‐mediated tumour–endothelial crosstalk

doi: 10.1002/ctm2.70861

Figure Lengend Snippet: FGF–FGFR‐mediated endothelial–tumour crosstalk. (A) Uniform manifold approximation and projection (UMAP) plots showing annotation of endothelial cell subpopulations. (B) Bubble plots displaying marker genes used for endothelial subpopulation annotation. (C) Pseudotime trajectory analysis of endothelial cells, showing differentiation progression. (D) Box plots comparing differentiation states among endothelial subpopulations. (E) CytoTRACE analysis depicting differentiation status across endothelial subpopulations. (F) Heatmap of branch‐dependent highly expressed genes along the endothelial pseudotime trajectory. (G) Circos‐style plot illustrating interaction strength between endothelial subpopulations and other major annotated cell types; arc thickness represents signal intensity. (H) Heatmap showing activation levels of the fibroblast growth factor (FGF)signalling pathway across endothelial subpopulations. (I) Cell–cell interaction heatmap displaying intercellular signalling activity. (J) NicheNet analysis showing ligand–receptor distribution between tumour cells and other cell types. (K) Expression distribution of FGFR1, FGFR2 and FGFR3 across different cell subclusters.

Article Snippet: The antibodies used and their catalogue numbers are as follows: SEMA3C (Novus Biologicals, Cat# MAB1728), CD31 (Servicebio, Cat# GB11063‐1), VWF (Servicebio, Cat# GB11020), FGFR2 (Proteintech, Cat# 13042‐1‐AP), PDGFRβ (Epizyme, Cat# P012976), VEGFR2 (Epizyme, Cat# P013400), CK7 (Biodragon, Cat# BD‐PM3054), Ki67 (Servicebio, Cat# GB111499 ) and EBF2 (Affinity, Cat# DF13398).

Techniques: Marker, Analysis, Activation Assay, Activity Assay, Expressing

Journal: Clinical and Translational Medicine

Article Title: Neuro‐associated SEMA3C + tumour cells promote gastric cancer peritoneal metastasis via EBF2–FGF7–FGFR2‐mediated tumour–endothelial crosstalk

doi: 10.1002/ctm2.70861

Figure Lengend Snippet: EBF2‐regulated tumour cell subpopulation mediates semaphorin 3C (SEMA3C) effects. (A) Bright‐field and haematoxylin and eosin (H&E) images showing interactions between tumour organoids and tumour‐associated endothelial cells (TECs) across different experimental groups (bright‐field scale bar: 100 µm; H&E scale bar: 20 µm). (B) Immunofluorescence images depicting the expression of relevant markers and the extent of organoid–endothelial cell interactions after co‐culture in different groups (scale bar: 30 µm). (C) Immunofluorescence images showing organoid–TEC interactions and marker expression across experimental conditions (scale bar: 30 µm). (D) pySCENIC analysis illustrating transcription factor activity within the SEMA3C + tumour cell subpopulation. (E) Tripartite plot displaying predicted target genes regulated by EBF2 in the SEMA3C + subpopulation. (F) Immunofluorescence images showing co‐staining of tumour cell subpopulation marker genes and predicted transcription factors under different experimental conditions (scale bar: 30 µm).

Article Snippet: The antibodies used and their catalogue numbers are as follows: SEMA3C (Novus Biologicals, Cat# MAB1728), CD31 (Servicebio, Cat# GB11063‐1), VWF (Servicebio, Cat# GB11020), FGFR2 (Proteintech, Cat# 13042‐1‐AP), PDGFRβ (Epizyme, Cat# P012976), VEGFR2 (Epizyme, Cat# P013400), CK7 (Biodragon, Cat# BD‐PM3054), Ki67 (Servicebio, Cat# GB111499 ) and EBF2 (Affinity, Cat# DF13398).

Techniques: Organoids, Immunofluorescence, Expressing, Co-culture Assay, Marker, Analysis, Activity Assay, Staining

Journal: Clinical and Translational Medicine

Article Title: Neuro‐associated SEMA3C + tumour cells promote gastric cancer peritoneal metastasis via EBF2–FGF7–FGFR2‐mediated tumour–endothelial crosstalk

doi: 10.1002/ctm2.70861

Figure Lengend Snippet: Semaphorin 3C (SEMA3C) drives tumour expansion via FGFR2‐mediated endothelial interaction. (A) Schematic diagram of the patient‐derived organoids‐based xenograft (PDOX) model construction and drug administration protocol. (B) Representative gross images of tumours from PDOX models in different experimental groups ( n = 5 mice per group). (C) Bar graph showing tumour weights across different groups. (D) Bar graph showing tumour volumes across different groups. (E) Immunofluorescence images showing the expression of relevant markers in tumour samples from different groups (scale bar: 50 µm). (F) Quantification of Ki67‐positive cells in tumour samples across experimental groups. (G) Quantification of CK7‐positive cells in tumour samples across experimental groups. (H) Quantification of VEGFR2 fluorescence intensity in tumour samples across groups. (I) Quantification of FGFR2‐positive cells in tumour samples across groups.

Article Snippet: The antibodies used and their catalogue numbers are as follows: SEMA3C (Novus Biologicals, Cat# MAB1728), CD31 (Servicebio, Cat# GB11063‐1), VWF (Servicebio, Cat# GB11020), FGFR2 (Proteintech, Cat# 13042‐1‐AP), PDGFRβ (Epizyme, Cat# P012976), VEGFR2 (Epizyme, Cat# P013400), CK7 (Biodragon, Cat# BD‐PM3054), Ki67 (Servicebio, Cat# GB111499 ) and EBF2 (Affinity, Cat# DF13398).

Techniques: Patient-Derived Organoids, Xenograft Assay, Immunofluorescence, Expressing, Fluorescence, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Neuro‐associated SEMA3C + tumour cells promote gastric cancer peritoneal metastasis via EBF2–FGF7–FGFR2‐mediated tumour–endothelial crosstalk

doi: 10.1002/ctm2.70861

Figure Lengend Snippet: Therapeutic targeting of the EBF2–FGF7–FGFR2 signalling axis. (A) Representative gross images showing tumour sizes in patient‐derived xenograft (PDX) models across different experimental groups ( n = 5 mice per group). (B) Bar graphs depicting tumour weights and volumes in different groups. (C) Immunofluorescence images showing the expression of relevant markers in tumour samples from different groups (scale bar: 50 µm).

Article Snippet: The antibodies used and their catalogue numbers are as follows: SEMA3C (Novus Biologicals, Cat# MAB1728), CD31 (Servicebio, Cat# GB11063‐1), VWF (Servicebio, Cat# GB11020), FGFR2 (Proteintech, Cat# 13042‐1‐AP), PDGFRβ (Epizyme, Cat# P012976), VEGFR2 (Epizyme, Cat# P013400), CK7 (Biodragon, Cat# BD‐PM3054), Ki67 (Servicebio, Cat# GB111499 ) and EBF2 (Affinity, Cat# DF13398).

Techniques: Derivative Assay, Xenograft Assay, Immunofluorescence, Expressing

Journal: Clinical and Translational Medicine

Article Title: Neuro‐associated SEMA3C + tumour cells promote gastric cancer peritoneal metastasis via EBF2–FGF7–FGFR2‐mediated tumour–endothelial crosstalk

doi: 10.1002/ctm2.70861

Figure Lengend Snippet: Schematic illustration summarising the key findings from single‐nucleus sequencing of gastric cancer peritoneal metastases. A tumour subpopulation with high semaphorin 3C (SEMA3C) expression was identified, which exhibits extensive interactions with tumour‐associated endothelial cells (TECs). TECs display high VEGFR2 expression, and transcriptional regulation by EBF2 in the tumour subpopulation leads to upregulation of FGF7. Tumour cells further interact with endothelial cells via the FGF7–FGFR2 signalling axis, promoting vascular remodelling, tumour proliferation and metastatic potential.

Article Snippet: The antibodies used and their catalogue numbers are as follows: SEMA3C (Novus Biologicals, Cat# MAB1728), CD31 (Servicebio, Cat# GB11063‐1), VWF (Servicebio, Cat# GB11020), FGFR2 (Proteintech, Cat# 13042‐1‐AP), PDGFRβ (Epizyme, Cat# P012976), VEGFR2 (Epizyme, Cat# P013400), CK7 (Biodragon, Cat# BD‐PM3054), Ki67 (Servicebio, Cat# GB111499 ) and EBF2 (Affinity, Cat# DF13398).

Techniques: Sequencing, Expressing

Journal: Journal of Clinical and Translational Hepatology

Article Title: Overexpression of RBM34 Promotes Tumor Progression and Correlates with Poor Prognosis of Hepatocellular Carcinoma

doi: 10.14218/jcth.2022.00166

Figure Lengend Snippet: Fig. 8. Suppression of RBM34 enhances sorafenib sensitivity in part by downregulating FGFR2. (A) qPCR assays validate the target genes of RBM34 in Huh7 cells. (B) Correlation between RBM34 and FGFR2 expression by bioinformatics analysis using the TCGA database. (C) FGFR2 protein expression in HCC cells was as- sayed in western blots. (D) Kaplan-Meier plots of overall survival in HCC patients with different RBM34 expression levels and sorafenib treatment. (E) Flow cytometry analysis of sorafenib-induced apoptosis in Huh7 cells with or without RBM34 expression. (F) Sorafenib treatment significantly inhibits AKT activity and not ERK activity in RBM34 knockout Huh7 cells compared with control cells. HCC, hepatocellular carcinoma; RBM, RNA-binding motif.

Article Snippet: Antibodies against RBM34 (#A10139), FGFR2 (#A19051), phospho-AKT1-S473 (#AP0140), AKT (#A18120) phosphoERK1/2 (#AP0472), ERK1/ERK2 (#A11116), HRP goat antimouse IgG (H+L) (#AS003), and HRP goat anti- rabbit IgG (#AS029) were purchased from Abclonal.

Techniques: Expressing, Western Blot, Flow Cytometry, Activity Assay, Knock-Out, Control, RNA Binding Assay

Journal: PloS one

Article Title: Differential roles of fibroblast growth factor receptors (FGFR) 1, 2 and 3 in the regulation of S115 breast cancer cell growth.

doi: 10.1371/journal.pone.0049970

Figure Lengend Snippet: Figure 1. FGFR1, 2 and 3 expression in FGFR-silenced S115 cells (sh cells). Cells were grown in DMEM containing 4% iFBS and 10 nM testosterone. RNA and protein were extracted from three independently cultured sub-confluent cell plates and analyzed using qRT-PCR and immunoblotting, respectively. A) FGFR1-3 mRNA expression in the five original FGFR-silenced cell pools (shR1B, shR1D, shR2IA, shR2ADG and shR3B) compared to the expression level in control cells (shLacZ). FGFR mRNA expression is normalized to cyclophilin B expression. The statistical differences between mRNA levels were tested by non-parametric Mann-Whitney U-test, * P,0.05, ** P,0.01. B) Overall relative FGFR mRNA expression level in sh cells. C) Relative FGFR1IgIIIb/c and FGFR2IgIIIb/c mRNA expression in cells that were chosen for further studies (shR1B, shR2IA and shR3B). D) FGFR protein levels in shR1B, shR2IA and shR3B cells. Immunoprecipitation of FGFR1 was performed using 150 mg of whole cell lysates. Detection of FGFR2 and FGFR3 by western blotting was performed using 20 mg of whole cell lysates. FGFR1 expression in shR1, shR2 and shR3 cells is normalized to the protein expression in shLacZ cells. FGFR2 and FGFR3 expression in shR1, shR2 and shR3 cells are normalized to b-actin expression and thereafter to expression in shLacZ. A representative immunoblot of three independent experiments is shown. doi:10.1371/journal.pone.0049970.g001

Article Snippet: FGFR2 Transfections The FGFR2 expression constructs SC112364 and SC111932 (here named pFGFR2IgIIIb and pFGFR2IgIIIc, respectively) were purchased from Origene (OriGene Technologies, Inc, Rockville, MD). shLacZ cells were transfected with FGFR2IgIIIb or FGFR2IgIIIc plasmids or with the vector control pCMV6-Neo using Lipofectamine 2000 (Invitrogen) according to the manufacturers’ instructions.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, Control, MANN-WHITNEY, Immunoprecipitation

Journal: PloS one

Article Title: Differential roles of fibroblast growth factor receptors (FGFR) 1, 2 and 3 in the regulation of S115 breast cancer cell growth.

doi: 10.1371/journal.pone.0049970

Figure Lengend Snippet: Figure 7. Overexpression of FGFR2IgIIIb and FGFR2IgIIIc in shLacZ cells. A) FGFR2 mRNA levels (relative to control-transfected cells (pCMV6-neo)) in FGFR2IgIIIb and FGFR2IgIIIc-transfected cells were analyzed by qRT-PCR. mRNA levels measured 24 h post-transfection are presented in a logarithmic scale. B) FGFR2 protein levels 24 h post-transfection shown by western blotting. C) Relative FGFR1 mRNA expression in FGFR2IgIIIb and FGFRIgIIIc-overexpressing cells 24 h post-transfection. The experiment was repeated twice with similar results. doi:10.1371/journal.pone.0049970.g007

Article Snippet: FGFR2 Transfections The FGFR2 expression constructs SC112364 and SC111932 (here named pFGFR2IgIIIb and pFGFR2IgIIIc, respectively) were purchased from Origene (OriGene Technologies, Inc, Rockville, MD). shLacZ cells were transfected with FGFR2IgIIIb or FGFR2IgIIIc plasmids or with the vector control pCMV6-Neo using Lipofectamine 2000 (Invitrogen) according to the manufacturers’ instructions.

Techniques: Over Expression, Control, Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: Microbiome

Article Title: Gardnerella vaginalis alters cervicovaginal epithelial cell function through microbe-specific immune responses.

doi: 10.1186/s40168-022-01317-9

Figure Lengend Snippet: Fig. 1 Co-localization of cervicovaginal epithelial cells with live G. vaginalis but not L. crispatus results in increased cell death. An in vitro live bacteria and host cervicovaginal co-culture model were created to study host microbial interactions in the CV space. Representative images of ectocervical (A, B), endocervical (C, D), and vaginal (E, F) epithelial cells interacting with L. crispatus (A, C, E) or G. vaginalis (B, D, F) are shown. Exposure of ectocervical (G), endocervical (H), and vaginal (I) cells to G. vaginalis but not L. crispatus results in dose-dependent cell death after 24 h. Values are mean ± SEM. Asterisks over the individual bars represent comparisons with control; asterisks over solid lines represent comparisons between treatment groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: Ectocervical (Ect/E6E7, ATCC # CRL-2614) (Ecto), endocervical (End1/E6E7, ATCC CRL-2615) (Endo), and vaginal (VK2/E6E7, ATCC CRL-2616) (VK2) human epithelial cell lines (American Type Culture Collection, Manassas, VA) were cultured in keratinocyte-serum-free media (K-SFM) supplemented with 0.1 ng/mL epidermal growth factor and 50 ug/mL bovine pituitary extract (Gibco, Life Technologies), 100 U/mL penicillin, and 100 μg/mL of streptomycin at 37 °C in a 5% CO2 humidified incubator.

Techniques: In Vitro, Bacteria, Co-Culture Assay, Control

Journal: Microbiome

Article Title: Gardnerella vaginalis alters cervicovaginal epithelial cell function through microbe-specific immune responses.

doi: 10.1186/s40168-022-01317-9

Figure Lengend Snippet: Fig. 2 Live G. vaginalis increases epithelial barrier permeability. Cell permeability was measured in ectocervical, endocervical, and vaginal epithelial cells after 24-h exposure to live bacteria (A, C, E) or bacteria-free supernatants (B, D, F) of L. crispatus or G. vaginalis. Bacterial growth media alone acted as a negative control for the bacteria-free supernatants tested. Cell permeability is expressed as fluorescence OD measurements from a fluorescent plate reader and is indicative of the movement of FITC-dextran from the top to the bottom insert of a transwell chamber system. Values are mean ± SEM. Asterisks over the individual bars represent comparisons with control; asterisks over solid lines represent comparisons between treatment groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: Ectocervical (Ect/E6E7, ATCC # CRL-2614) (Ecto), endocervical (End1/E6E7, ATCC CRL-2615) (Endo), and vaginal (VK2/E6E7, ATCC CRL-2616) (VK2) human epithelial cell lines (American Type Culture Collection, Manassas, VA) were cultured in keratinocyte-serum-free media (K-SFM) supplemented with 0.1 ng/mL epidermal growth factor and 50 ug/mL bovine pituitary extract (Gibco, Life Technologies), 100 U/mL penicillin, and 100 μg/mL of streptomycin at 37 °C in a 5% CO2 humidified incubator.

Techniques: Permeability, Bacteria, Negative Control, Fluorescence, Control

Journal: Microbiome

Article Title: Gardnerella vaginalis alters cervicovaginal epithelial cell function through microbe-specific immune responses.

doi: 10.1186/s40168-022-01317-9

Figure Lengend Snippet: Fig. 6 Immune profile of cervicovaginal epithelial cells identifies cytokines mediated by activation of TLR2. Blocking the TLR2 receptor alters the immune cytokines/chemokines released from ectocervical (A), endocervical (B), and vaginal cells (C) after exposure to live L. crispatus and G. vaginalis. Heat map depicts fold change by color and p-value by asterisks. Fold change was calculated between the non-treated (NTC) control and live bacteria alone or live bacteria plus anti-TLR2 antibody. p-value is based on pg/ml values. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: Ectocervical (Ect/E6E7, ATCC # CRL-2614) (Ecto), endocervical (End1/E6E7, ATCC CRL-2615) (Endo), and vaginal (VK2/E6E7, ATCC CRL-2616) (VK2) human epithelial cell lines (American Type Culture Collection, Manassas, VA) were cultured in keratinocyte-serum-free media (K-SFM) supplemented with 0.1 ng/mL epidermal growth factor and 50 ug/mL bovine pituitary extract (Gibco, Life Technologies), 100 U/mL penicillin, and 100 μg/mL of streptomycin at 37 °C in a 5% CO2 humidified incubator.

Techniques: Activation Assay, Blocking Assay, Control, Bacteria

Journal: Microbiome

Article Title: Gardnerella vaginalis alters cervicovaginal epithelial cell function through microbe-specific immune responses.

doi: 10.1186/s40168-022-01317-9

Figure Lengend Snippet: Fig. 7 Blocking the TLR2 receptor does not mitigate G. vaginalis-induced increases in cell permeability. Cell permeability was measured in ectocervical (A), endocervical (B), and vaginal (C) epithelial cells after pretreatment with the anti-TLR2 antibody and subsequent exposure to live bacteria. G. vaginalis-induced cell permeability was unchanged after blocking the TLR2 receptor. Values are mean ± SEM

Article Snippet: Ectocervical (Ect/E6E7, ATCC # CRL-2614) (Ecto), endocervical (End1/E6E7, ATCC CRL-2615) (Endo), and vaginal (VK2/E6E7, ATCC CRL-2616) (VK2) human epithelial cell lines (American Type Culture Collection, Manassas, VA) were cultured in keratinocyte-serum-free media (K-SFM) supplemented with 0.1 ng/mL epidermal growth factor and 50 ug/mL bovine pituitary extract (Gibco, Life Technologies), 100 U/mL penicillin, and 100 μg/mL of streptomycin at 37 °C in a 5% CO2 humidified incubator.

Techniques: Blocking Assay, Permeability, Bacteria

Journal: PLoS ONE

Article Title: Receptor Tyrosine Kinases Activate Canonical WNT/β-Catenin Signaling via MAP Kinase/LRP6 Pathway and Direct β-Catenin Phosphorylation

doi: 10.1371/journal.pone.0035826

Figure Lengend Snippet: (A) RCS cells were transfected with wt FGFR3 or activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650M, K650E), and analyzed for the indicated molecules by WB 48 hours later. The levels of ERK phosphorylation vary among the tested mutants, reflecting the different strength of FGFR3 activation by each particular mutation . K508M - kinase inactive FGFR3 mutant. GFP and empty vectors serve as transfection controls. (B) LRP6 phosphorylation at Thr1572 caused by highly activating FGFR3 mutants R248C and K650E. (C) Cells were transfected with the indicated FGFR3 vectors together with Topflash reporter vectors, treated with WNT3a and analyzed for luciferase activity. Data represent an average from three transfections (each measured twice), with the indicated standard deviations. A logarithmic scale of the y -axis is necessary to express the massive Topflash activation in WNT3a-treated cells expressing activating FGFR3 mutants (* p <0.001; Student’s t -test; compared to wt FGFR3). Results are representative of four experiments. (D) Cells were transfected with wt FGFR2 or activating FGFR2 mutants (S252W, P253R, C342R, C342Y, Y375C), and analyzed for the indicated molecules by WB. Note the significant ERK and LRP6 phosphorylation caused by C342R, C342Y and Y375C mutants, which correlates with increased basal (E; upper graph) and WNT3a-induced (E; lower graph) β-catenin activity, evidenced by Topflash experiment. Results are representative for three experiments (* p <0.001; Student’s t -test; compared to wt FGFR2).

Article Snippet: For the recombinant RTK kinase assays, the reactions were carried-out with 400 ng of recombinant FGFR2, FGFR3, TRKA or EGFR (SignalChem, Richmond, Canada) and 400 ng of recombinant β-catenin (Abcam, Cambridge, MA, USA) in 50 μl of kinase buffer in the presence of 50 μM ATP for 60 minutes at 30°C.

Techniques: Transfection, Phospho-proteomics, Activation Assay, Mutagenesis, Luciferase, Activity Assay, Expressing

Journal: PLoS ONE

Article Title: Receptor Tyrosine Kinases Activate Canonical WNT/β-Catenin Signaling via MAP Kinase/LRP6 Pathway and Direct β-Catenin Phosphorylation

doi: 10.1371/journal.pone.0035826

Figure Lengend Snippet: (A) RCS cells were treated for indicated times with FGF2 (10 ng/ml) in the presence of heparin (1 µg/ml), and analyzed for β-catenin phosphorylation at Tyr142 by WB (arrow). (B) HEK293 cells were transfected with wt FGFR2 or its activating mutant Y375C, and analyzed for indicated molecules 48 hours later. Note the increased β-catenin phosphorylation at Tyr142 (arrow). (C) Active recombinant FGFR3, FGFR2, TRKA and EGFR were subjected to a cell-free kinase assay with recombinant β-catenin as a substrate. Samples with ATP or kinase omitted serve as controls for kinase reaction.

Article Snippet: For the recombinant RTK kinase assays, the reactions were carried-out with 400 ng of recombinant FGFR2, FGFR3, TRKA or EGFR (SignalChem, Richmond, Canada) and 400 ng of recombinant β-catenin (Abcam, Cambridge, MA, USA) in 50 μl of kinase buffer in the presence of 50 μM ATP for 60 minutes at 30°C.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Recombinant, Kinase Assay