Journal: International journal of molecular sciences

Article Title: Using Cancer-Associated Fibroblasts as a Shear-Wave Elastography Imaging Biomarker to Predict Anti-PD-1 Efficacy of Triple-Negative Breast Cancer.

doi: 10.3390/ijms26083525

Figure Lengend Snippet: Figure 2. Woundhealing CAF high-proportion tumors exhibit greater immune suppression. (A) Gene ontology (GO) enrichment analysis revealed the downregulation of the top 30 pathways between high- and low-proportion groups. Focus pathways were marked in red. (B) Gene set enrichment analysis (GSEA) of immune response, T cell activation, programmed cell death protein 1 (PD-L1) expression, and PD-1 checkpoint pathway in cancer between high- and low-proportion groups. (C) Immunofluorescence staining of CD8 (pink) expression distribution in tumor tissues, scale bar: 100 µm. (D,E) Heatmap and GSEA showed one-carbon metabolism and related pathways in high- and low-proportion groups. (F) Various forms of metabolism in high- and low-proportion groups. Methylation was marked in red. (G) Addmodulescore in pathway (one carbon metabolic process and one carbon compound transport) based on gene expression of different CAF subtypes, focus subtypes marked in red, and the red dashed line represents the median value of WH CAFs as the reference line. (H) The immunofluorescence co-staining image of serine hydroxymethyltransferase (SHMT) (red) and CD8 (green), scale bar: 2000 µm (low magnification), 100 µm (high magnification). (I) GSEA of FGFR signaling pathway in high and low groups. (J) FGFR2 and PD-L1 protein expres- sion in high- and low-proportion groups. (K) Immunofluorescence staining of fibroblast growth factor receptor2 (FGFR2) (red) between high- and low-proportion groups, and quantitative results, scale bar: 2000 µm (low magnification), 100 µm (high magnification). (n = 5 independent samples) (1: Extracellular Matrix CAFs; 2: Matrix CAFs 1; 3: Development CAFs; 4: Antigen presentation CAFs 1; 5: Antigen presentation CAFs 2; 6: Wound healing CAFs; 7: Vascular CAFs; 8: Inflammatory CAFs; 9: Matrix CAFs 2). Data expressed as mean ± S.E.M; statistical comparisons were performed using Student’s t test.

Article Snippet: The primary antibodies used for IF staining include VTN antibody (Proteintech, Wuhan, China, 66398-1-lg), FGFR2 antibody (Proteintech, Wuhan, China, 13042-1-AP), CD8a antibody (Proteintech, Wuhan, China, 29896-1-AP), and SHMT1 antibody (Proteintech, Wuhan, China, 67963-1-Ig).

Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Methylation, Gene Expression, Immunopeptidomics

Journal: International journal of molecular sciences

Article Title: Using Cancer-Associated Fibroblasts as a Shear-Wave Elastography Imaging Biomarker to Predict Anti-PD-1 Efficacy of Triple-Negative Breast Cancer.

doi: 10.3390/ijms26083525

Figure Lengend Snippet: Figure 4. FGFR inhibitor potentiates immune checkpoint inhibitors (ICIs) in WH CAF high-level tumors. (A) Schematic of the experimental protocol. SWE: shear wave elastography. (B) Representa- tive SWE images of tumors in high-level and low-level groups at the end of volume measurement. (C) Mean stiffness value of tumors in high-level and low-level groups (WH high-level group: n = 27, WH low-level group: n = 14). (D) Tumor volume curves in different groups during the 15-day monitoring period (Group: WH CAFs high level, WH CAFs high level with Anti-PD-1, WH CAFs high level with Anti-PD-1+Erdafitinib: n = 9; group: WH CAFs low level, WH CAFs low level with Anti-PD-1: n = 5; group: WH CAFs low level with Anti-PD-1+Erdafitinib: n = 4). (E) Comparison of immunohistochemical results of CD8 and CD3 in high- and low-level groups before and after Anti- PD-1+Erdafitinib treatment, scale: 100 µm. (F) Immunofluorescence staining and quantization results of FGFR2 and VTN proteins in WH CAFs high-level group before and after Anti-PD-1+Erdafitinib treatment, scale bar: 2000 µm (low magnification), 100 µm (high magnification). Data expressed as Mean ± S.E.M; statistical comparisons were performed using Student’s t test.

Article Snippet: The primary antibodies used for IF staining include VTN antibody (Proteintech, Wuhan, China, 66398-1-lg), FGFR2 antibody (Proteintech, Wuhan, China, 13042-1-AP), CD8a antibody (Proteintech, Wuhan, China, 29896-1-AP), and SHMT1 antibody (Proteintech, Wuhan, China, 67963-1-Ig).

Techniques: Shear, Comparison, Immunohistochemical staining, Immunofluorescence, Staining

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

Journal: International Journal of Clinical and Experimental Pathology

Article Title: CXCL5 contributes to the tumorigenicity of cervical cancer and is post-transcriptionally regulated by miR-577

doi:

Figure Lengend Snippet: CXCL5 is up-regulated in cervical cancer tissues and cell lines. IHC staining was performed to analyze CXCL5 positive staining in cervical cancer tissues and para-carcinoma tissues (A: left: magnification 40×; right: magnification 200×) and quantitative analysis (B). Western blot was used to measure CXCL5 protein expression in human normal cervical squamous cell line Ect1/E6E7 and cervical cancer cell lines Hela and SiHa (C and D). *P < 0.05. CC, cervical cancer.

Article Snippet: Human normal cervical squamous cell line Ect1/E6E7 (CHI Scientific, Inc., Maynard, MA, USA) and cervical cancer cell lines Hela and SiHa (ATCC, USA) were maintained in DMEM; (Gibco, USA) containing 10% FBS (Gibco, USA) with 5% CO 2 at 37°C. miR-Con, miR-577 mimics, miR-577-Mut, and overexpressed CXCL5 plasmids were purchased from GenePharm (Shanghai, China) and transfected into Hela and SiHa cells using lipofectamine 2000 (Invitrogen).

Techniques: Immunohistochemistry, Staining, Western Blot, Expressing