Journal: Journal of Translational Medicine

Article Title: Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology

doi: 10.1186/s12967-024-05159-x

Figure Lengend Snippet: Construction of FGFR4 nanoparticles and immunization of alpaca. a Schematic diagram of expression vector encoding FGFR4 antigen’s extracellular domain proteins and ferritin. b Schematic diagram of generation of FGFR4-HPF nanoparticles. c Process of immunizing alpaca with FGFR4-HPF nanoparticles. d Immunized alpaca serum ELISA

Article Snippet: Mouse anti-human PE-FGFR4 antibody (Abcam, 4FR6D3) was used for detection of FGFR4.

Techniques: Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology

doi: 10.1186/s12967-024-05159-x

Figure Lengend Snippet: Construction of electroporation bacteria library and screening of anti-FGFR4 Nbs. a Monoclonal sequencing analysis and genetic evolutionary tree analysis of the electroporation library. Sequencing of 72 monoclonal clones identified 69 positive clones with insertion of single Nb sequences. b Proportion of Nb-displaying phages capable of binding to FGFR4 after screening. Left: output of first round; middle: input of second round; right: output of second round. c Monoclonal phage ELISA with FGFR4. Positive clones: 1, 9, 13, 14, 19. 20, 22, 23. d Identification of monoclonal phages capable of binding to Huh7 cells by flow cytometry. The NC phages uncapable of binding Huh7 cells were used as the control. Clones with positive fluorescence: 1 (anti-FGFR4 Nb1), 14 (anti-FGFR4 Nb2)

Article Snippet: Mouse anti-human PE-FGFR4 antibody (Abcam, 4FR6D3) was used for detection of FGFR4.

Techniques: Electroporation, Bacteria, Sequencing, Clone Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence

Journal: Journal of Translational Medicine

Article Title: Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology

doi: 10.1186/s12967-024-05159-x

Figure Lengend Snippet: Functional validation of the screened Nbs in vitro . a Molecular docking models of Nbs with FGFR4. The structure of light blue represents antigen, the structure of dark blue represents Nbs, the interface regions of red and green represent the region of the Nb in contact with antigen, ΔG denotes the free energy of binding. b Schematic diagram of expression vector encoding Nb-Fc. c Detection of the binding ability of Nbs by antibody-antigen binding ELISA. Nc-Linker-Fc: non FGFR4 targeting control nanobody-Linker-Fc antibody. Data were analyzed by the Student’s t-test. d Detection of the binding ability of Nbs by antibody gradient dilution ELISA. Nc-Linker-Fc: non FGFR4 targeting control nanobody-Linker-Fc antibody. Data were analyzed by two-way ANOVA. e Detection of the binding affinity of Nbs by SPR assay. f Evaluation the specificity of Nbs by antibody-antigen binding ELISA. Data were analyzed by one-way ANOVA. The experiments were performed independently in triplicate. Data are expressed as mean ± SEM. **** p < 0.0001

Article Snippet: Mouse anti-human PE-FGFR4 antibody (Abcam, 4FR6D3) was used for detection of FGFR4.

Techniques: Functional Assay, In Vitro, Binding Assay, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, SPR Assay

Journal: Journal of Translational Medicine

Article Title: Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology

doi: 10.1186/s12967-024-05159-x

Figure Lengend Snippet: Construction of Nb-derived CAR-T cells and functional validation of Nb-derived CAR-T cells anti-tumor in vitro . a Schematic diagram of lentivirus plasmid encoding anti-FGFR4 Nb-CAR. b Validation of cytotoxicity of Nb-derived CAR-T cells against Huh7 cells in vitro by LDH assay. NC-T: T cells transduced with empty lentivirus vector. c Validation of cytotoxicity of Nb-derived CAR-T cells against BXPC3 cells in vitro by LDH assay. NC-T: T cells transduced with empty lentivirus vector. d Validation of the cytokine secretion functions of Nb-derived CAR-T cells against Huh7 cells in vitro by ELISA. Mock: T cells transduced with empty lentivirus vector. e Validation of the IFN-γ secretion functions of Nb-derived CAR-T cells against Huh7 cells in vitro by ELISPOT. Mock: T cells transduced with empty lentivirus vector. Data were analyzed by two-way ANOVA. The experiments were performed independent biological replicates (N = 3). Data are expressed as mean ± SEM. Ns: p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Mouse anti-human PE-FGFR4 antibody (Abcam, 4FR6D3) was used for detection of FGFR4.

Techniques: Derivative Assay, Functional Assay, In Vitro, Plasmid Preparation, Lactate Dehydrogenase Assay, Transduction, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 1. High FGFR4 expression is associated with the oxidative stress pathway in gastric cancer. A) The Ridge plot by Gene set enrichment analysis (GSEA) was performed using FGFR4high expression samples, compared with FGFR4low expression samples in the TCGA and eight GEO databases (GSE66229, GSE15459, GSE26901, GSE13861, GSE110875, GSE34942, GSE100935 and GSE30727).Hallmark gene sets were downloaded from MSigDB (https://www.gsea-msigdb.org/). B) Oxidative phosphorylation gene set and reactive oxygen species pathway were significantly enriched in high FGFR4 expression samples in the TCGA cohort. C) Representative images of CM-H2DCFDA immunofluorescent staining showing higher ROS levels in MKN28 cells infected with H. pylori J166 or 7.13 strains (3h). ROS levels were significantly increased following FGFR4 knockdown. D) Quantification of CM-H2DCFDA positive staining in at least two hundred cells from three images is presented as a percentage in the right panel. Data are graphed with mean ± SEM. E) Immunofluorescence staining of 8-oxoguanine demonstrates a significant increase in oxidative DNA damage after infection with H. pylori. This increase was significantly enhanced following FGFR4 knockdown. F) quantification of 8-oxo guanine immunofluorescent staining as in D. Data are graphed with mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Next, the tissue sections were incubated with two primary antibodies, rabbit NRF2 from Novus Biologicals and mouse FGFR4 from Santa Cruz Biotechnology, diluted in blocking buffer (1:200) overnight at 4 ◦C.

Techniques: Expressing, Phospho-proteomics, Staining, Infection, Knockdown, Immunofluorescence

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 2. Positive correlation between FGFR4 and NRF2 in gastric cancer. A) Pearson’s correlation analysis between FGFR4 mRNA level and single-sample gene set enrichment analysis (ssGSEA) scores for NRF2 targets’ signature in the TCGA cohort. B) GSEA was performed using samples with FGFR4high expression compared to samples with FGFR4low expression in TCGA cohort. NRF2 signature [49] was significantly enriched in FGFR4high expression samples. C) Immunofluorescence analysis shows an increase of NRF2 nuclear staining (green) and FGFR4 expression (red) in TFF1-KO mouse neoplastic gastric tissues, as compared to normal gastric tissues from the TFF1-WT mice (scale of 10 μm is shown in the merged image). The arrows point to nuclei. The right panels show bright field image of H&E staining. D) Western blot analysis demonstrates an increase of p-FGFR4 (Y642), FGFR4, NRF2 and HO1 protein levels in neoplastic gastric tissues (KO), as compared to normal tissue samples (WT) from mice. RT-qPCR analysis of Fgfr4, Fgf15 and Ho1 mRNA expression in gastric tissues of TFF1-KO mouse as compared to TFF1-WT.*P < 0.05, **P < 0.01 and ***P < 0.001 is considered significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Next, the tissue sections were incubated with two primary antibodies, rabbit NRF2 from Novus Biologicals and mouse FGFR4 from Santa Cruz Biotechnology, diluted in blocking buffer (1:200) overnight at 4 ◦C.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 3. FGFR4 knockdown reduces NRF2 protein and transcription activity levels. A) Western blot shows an increase of FGFR4, NRF2 and HO1 proteins after H. pylori infection (3h) as compared to uninfected negative control (NC). FGFR4 siRNA knockdown abrogated this increase. B) Immunofluorescence assay demonstrates an increase of NRF2 nuclear staining in H. pylori Infected cells. This increase was abolished with FGFR4 siRNA knockdown. C) Quantification of nuclear NRF2-positive staining in at least 200 cells from three images is presented as a percentage in the right panel. Data are graphed with mean ± SEM. D) The NRF2 transcriptional activity was measured by the ARE luciferase reporter assay following infection with H. pylori in MKN28 cells. H. pylori induced the activity of the reporter, whereas FGFR4 siRNA knockdown reversed this effect. The luciferase reporter activity values were normalized to β-gal expression levels and are represented as luciferase activity relative to control. E-F) RT-qPCR of FGF19 (E) and HO1 (F) following infection with H. pylori (3h) in MKN28 cells transfected with control siRNA or FGFR4 siRNA. ***P < 0.001.

Article Snippet: Next, the tissue sections were incubated with two primary antibodies, rabbit NRF2 from Novus Biologicals and mouse FGFR4 from Santa Cruz Biotechnology, diluted in blocking buffer (1:200) overnight at 4 ◦C.

Techniques: Knockdown, Activity Assay, Western Blot, Infection, Negative Control, Immunofluorescence, Staining, Luciferase, Reporter Assay, Expressing, Control, Quantitative RT-PCR, Transfection

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 4. NRF2 nuclear expression is associated with H. pylori infection and tumorigenesis in mouse gastric tissues. A) Immunofluorescence analysis shows an increase of NRF2 (green) nuclear staining (arrow heads) and FGFR4 expression (red; arrow heads) in wild-type mice (C57/B6) gastric tissues after H. pylori (PMSS1 strain) infection for four weeks (scale of 10 μm is shown in the merged image). The right panels show bright field images of H&E staining. The arrowheads point to the nuclei. B) Western blot analysis demonstrates an increase in the protein levels of p-FGFR4 (Y642), FGFR4, NRF2 and HO1 in gastric tissues of mice infected with PMSS1 as compared to uninfected mice. C) RT-qPCR analysis of Fgfr4, Fgf15 and Ho1 mRNA expression in gastric tissues of mice infected with PMSS1 as compared to uninfected mice. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Next, the tissue sections were incubated with two primary antibodies, rabbit NRF2 from Novus Biologicals and mouse FGFR4 from Santa Cruz Biotechnology, diluted in blocking buffer (1:200) overnight at 4 ◦C.

Techniques: Expressing, Infection, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 5. FGFR4 inhibition decreases NRF2 in vitro and in vivo. A) Western blot analysis in MKN28 cells. Treatment with FGFR4 inhibitor (H3B-6527) abrogates H. pylori (7.13)-mediated increase in NRF2. B) The ARE luciferase reporter assay was used as a measure of NRF2 transcriptional activity under similar conditions as in B, demonstrating a significant induction of luciferase activity by H. pylori (7.13) infection, an effect that was abolished with the H3B-6527 inhibitor. The luciferase reporter activity values were normalized to β-gal expression levels and are represented as luciferase activity relative to control. C) Immunofluorescence analysis shows an increase of NRF2 (green) nuclear staining and FGFR4 expression (red) in wild-type mice (C57/B6) gastric tissues after four weeks of infection with H. pylori (PMSS1); nuclear localization of NRF2 was abolished after treatment with H3B-6527 (H3B); arrows point to nuclei. The right panels show bright field images of H&E staining. D) Western blot analysis of mouse gastric tissues shows an increase of p-FGFR4 (Y642), FGFR4 and NRF2 in infected mice; this increase was abolished after treatment with H3B-6527. E) RT-qPCR analysis of Fgfr4 and Ho1 mRNA expression in gastric tissues of mice infected with PMSS1 and treated or not with H3B-6527 (H3B) and compared to uninfected mice. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Next, the tissue sections were incubated with two primary antibodies, rabbit NRF2 from Novus Biologicals and mouse FGFR4 from Santa Cruz Biotechnology, diluted in blocking buffer (1:200) overnight at 4 ◦C.

Techniques: Inhibition, In Vitro, In Vivo, Western Blot, Luciferase, Reporter Assay, Activity Assay, Infection, Expressing, Control, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 6. FGFR4 induces P62 to promote NRF2 protein stability. A) Western blot analysis using MKN28 cells uninfected or infected with H. pylori (7.13, 3h). Infected cells show high levels of p-FGFR4 (Y642), FGFR4, NRF2, p-P62 (S349) and P62. These changes were reversed upon FGFR4 siRNA knockdown. The relative intensity ratios of NRF2/β-Actin, p-P62/β-Actin, and P62/β-Actin were calculated by ImageJ software and shown on the right panel. The results are expressed as mean ± SEM of at least three independent experiments. B) Western blot analysis following treatment of MKN28 cells with FGF19 (30 min). This stimulation led to increases in p- FGFR4 (Y642), FGFR4, NRF2, p-P62 (S349) and P62. These changes were reversed with FGFR4 siRNA knockdown. The Bar graphs show the relative intensity ratios of NRF2/β-Actin, p-P62/β-Actin, and P62/β-Actin were calculated and shown on the right panel. The results are expressed as mean ± SEM of at least three inde pendent experiments. C-D) Western blot showing cycloheximide chase assay of NRF2 using FGFR4 siRNA (C) or P62 siRNA (D) at the indicated time points, following H. pylori infection (3h). NRF2 protein stability was reduced following FGFR4 or P62 knockdown. The half-life time (t1/2) of NRF2 was calculated using GraphPad Prism software and plotted on the right side of panels C and D, respectively. *P < 0.05, **P < 0.001.

Article Snippet: Next, the tissue sections were incubated with two primary antibodies, rabbit NRF2 from Novus Biologicals and mouse FGFR4 from Santa Cruz Biotechnology, diluted in blocking buffer (1:200) overnight at 4 ◦C.

Techniques: Western Blot, Infection, Knockdown, Software

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 7. FGFR4, P62, and KEAP1 coexist in the same protein complex. A-B) Immunoprecipitation and western blot analysis following P62 pulldown (A) or FGFR4 pulldown (B) using MKN28 cells infected with H. pylori 7.13 (3 h). Immunoprecipitations and their corresponding input samples were subjected to immunoblotting with P62 FGFR4 Keap1 and NRF2 antibodies. The infection was confirmed using CagA antibody and equal amounts of protein loading were confirmed in the input samples using GAPDH antibody. C) Proximity ligation assay was performed in MKN28 cells transfected with control or FGFR4 siRNA and infected with H. pylori (7.13). The presence of red signals indicates positive ligation and proximity of the proteins, indicative of interaction. Using FGFR4 and P62 antibodies, the results indicated the presence of FGFR4-P62 interaction (red signals) following H. pylori infection (left upper panel). This interaction was not detected with FGFR4 siRNA (middle upper panel). The lower panels display the negative control for PLA background reaction. Control cells were transfected with Ctrl siRNA, infected with H. pylori and probed with a single antibody against FGFR4 (lower left panel) or P62 (lower right panel). The upper right panel displays a negative control for the PLA background with no antibody. Maximum intensity projection is presented on the right and lower sides of each image. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Next, the tissue sections were incubated with two primary antibodies, rabbit NRF2 from Novus Biologicals and mouse FGFR4 from Santa Cruz Biotechnology, diluted in blocking buffer (1:200) overnight at 4 ◦C.

Techniques: Immunoprecipitation, Western Blot, Infection, Proximity Ligation Assay, Transfection, Control, Ligation, Negative Control

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 9. A schematic diagram illustrating the role of FGFR4 in activating and stabilizing NRF2. Exposure of cells to H. pylori infection induces oxidative stress and FGFR4 expression. FGFR4 binds to P62. The FGFR4-P62 complex binds to KEAP1 in NRF2 degradation complex, competing with binding of KEAP1 top NRF2. NRF2 escapes degradation, accumulates, and translocates to the nucleus to induce transcription of antioxidant response genes.

Article Snippet: Next, the tissue sections were incubated with two primary antibodies, rabbit NRF2 from Novus Biologicals and mouse FGFR4 from Santa Cruz Biotechnology, diluted in blocking buffer (1:200) overnight at 4 ◦C.

Techniques: Infection, Expressing, Binding Assay

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 1. High FGFR4 expression is associated with the oxidative stress pathway in gastric cancer. A) The Ridge plot by Gene set enrichment analysis (GSEA) was performed using FGFR4high expression samples, compared with FGFR4low expression samples in the TCGA and eight GEO databases (GSE66229, GSE15459, GSE26901, GSE13861, GSE110875, GSE34942, GSE100935 and GSE30727).Hallmark gene sets were downloaded from MSigDB (https://www.gsea-msigdb.org/). B) Oxidative phosphorylation gene set and reactive oxygen species pathway were significantly enriched in high FGFR4 expression samples in the TCGA cohort. C) Representative images of CM-H2DCFDA immunofluorescent staining showing higher ROS levels in MKN28 cells infected with H. pylori J166 or 7.13 strains (3h). ROS levels were significantly increased following FGFR4 knockdown. D) Quantification of CM-H2DCFDA positive staining in at least two hundred cells from three images is presented as a percentage in the right panel. Data are graphed with mean ± SEM. E) Immunofluorescence staining of 8-oxoguanine demonstrates a significant increase in oxidative DNA damage after infection with H. pylori. This increase was significantly enhanced following FGFR4 knockdown. F) quantification of 8-oxo guanine immunofluorescent staining as in D. Data are graphed with mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Expressing, Phospho-proteomics, Staining, Infection, Knockdown, Immunofluorescence

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 2. Positive correlation between FGFR4 and NRF2 in gastric cancer. A) Pearson’s correlation analysis between FGFR4 mRNA level and single-sample gene set enrichment analysis (ssGSEA) scores for NRF2 targets’ signature in the TCGA cohort. B) GSEA was performed using samples with FGFR4high expression compared to samples with FGFR4low expression in TCGA cohort. NRF2 signature [49] was significantly enriched in FGFR4high expression samples. C) Immunofluorescence analysis shows an increase of NRF2 nuclear staining (green) and FGFR4 expression (red) in TFF1-KO mouse neoplastic gastric tissues, as compared to normal gastric tissues from the TFF1-WT mice (scale of 10 μm is shown in the merged image). The arrows point to nuclei. The right panels show bright field image of H&E staining. D) Western blot analysis demonstrates an increase of p-FGFR4 (Y642), FGFR4, NRF2 and HO1 protein levels in neoplastic gastric tissues (KO), as compared to normal tissue samples (WT) from mice. RT-qPCR analysis of Fgfr4, Fgf15 and Ho1 mRNA expression in gastric tissues of TFF1-KO mouse as compared to TFF1-WT.*P < 0.05, **P < 0.01 and ***P < 0.001 is considered significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 3. FGFR4 knockdown reduces NRF2 protein and transcription activity levels. A) Western blot shows an increase of FGFR4, NRF2 and HO1 proteins after H. pylori infection (3h) as compared to uninfected negative control (NC). FGFR4 siRNA knockdown abrogated this increase. B) Immunofluorescence assay demonstrates an increase of NRF2 nuclear staining in H. pylori Infected cells. This increase was abolished with FGFR4 siRNA knockdown. C) Quantification of nuclear NRF2-positive staining in at least 200 cells from three images is presented as a percentage in the right panel. Data are graphed with mean ± SEM. D) The NRF2 transcriptional activity was measured by the ARE luciferase reporter assay following infection with H. pylori in MKN28 cells. H. pylori induced the activity of the reporter, whereas FGFR4 siRNA knockdown reversed this effect. The luciferase reporter activity values were normalized to β-gal expression levels and are represented as luciferase activity relative to control. E-F) RT-qPCR of FGF19 (E) and HO1 (F) following infection with H. pylori (3h) in MKN28 cells transfected with control siRNA or FGFR4 siRNA. ***P < 0.001.

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Knockdown, Activity Assay, Western Blot, Infection, Negative Control, Immunofluorescence, Staining, Luciferase, Reporter Assay, Expressing, Control, Quantitative RT-PCR, Transfection

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 4. NRF2 nuclear expression is associated with H. pylori infection and tumorigenesis in mouse gastric tissues. A) Immunofluorescence analysis shows an increase of NRF2 (green) nuclear staining (arrow heads) and FGFR4 expression (red; arrow heads) in wild-type mice (C57/B6) gastric tissues after H. pylori (PMSS1 strain) infection for four weeks (scale of 10 μm is shown in the merged image). The right panels show bright field images of H&E staining. The arrowheads point to the nuclei. B) Western blot analysis demonstrates an increase in the protein levels of p-FGFR4 (Y642), FGFR4, NRF2 and HO1 in gastric tissues of mice infected with PMSS1 as compared to uninfected mice. C) RT-qPCR analysis of Fgfr4, Fgf15 and Ho1 mRNA expression in gastric tissues of mice infected with PMSS1 as compared to uninfected mice. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Expressing, Infection, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 5. FGFR4 inhibition decreases NRF2 in vitro and in vivo. A) Western blot analysis in MKN28 cells. Treatment with FGFR4 inhibitor (H3B-6527) abrogates H. pylori (7.13)-mediated increase in NRF2. B) The ARE luciferase reporter assay was used as a measure of NRF2 transcriptional activity under similar conditions as in B, demonstrating a significant induction of luciferase activity by H. pylori (7.13) infection, an effect that was abolished with the H3B-6527 inhibitor. The luciferase reporter activity values were normalized to β-gal expression levels and are represented as luciferase activity relative to control. C) Immunofluorescence analysis shows an increase of NRF2 (green) nuclear staining and FGFR4 expression (red) in wild-type mice (C57/B6) gastric tissues after four weeks of infection with H. pylori (PMSS1); nuclear localization of NRF2 was abolished after treatment with H3B-6527 (H3B); arrows point to nuclei. The right panels show bright field images of H&E staining. D) Western blot analysis of mouse gastric tissues shows an increase of p-FGFR4 (Y642), FGFR4 and NRF2 in infected mice; this increase was abolished after treatment with H3B-6527. E) RT-qPCR analysis of Fgfr4 and Ho1 mRNA expression in gastric tissues of mice infected with PMSS1 and treated or not with H3B-6527 (H3B) and compared to uninfected mice. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Inhibition, In Vitro, In Vivo, Western Blot, Luciferase, Reporter Assay, Activity Assay, Infection, Expressing, Control, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 6. FGFR4 induces P62 to promote NRF2 protein stability. A) Western blot analysis using MKN28 cells uninfected or infected with H. pylori (7.13, 3h). Infected cells show high levels of p-FGFR4 (Y642), FGFR4, NRF2, p-P62 (S349) and P62. These changes were reversed upon FGFR4 siRNA knockdown. The relative intensity ratios of NRF2/β-Actin, p-P62/β-Actin, and P62/β-Actin were calculated by ImageJ software and shown on the right panel. The results are expressed as mean ± SEM of at least three independent experiments. B) Western blot analysis following treatment of MKN28 cells with FGF19 (30 min). This stimulation led to increases in p- FGFR4 (Y642), FGFR4, NRF2, p-P62 (S349) and P62. These changes were reversed with FGFR4 siRNA knockdown. The Bar graphs show the relative intensity ratios of NRF2/β-Actin, p-P62/β-Actin, and P62/β-Actin were calculated and shown on the right panel. The results are expressed as mean ± SEM of at least three inde pendent experiments. C-D) Western blot showing cycloheximide chase assay of NRF2 using FGFR4 siRNA (C) or P62 siRNA (D) at the indicated time points, following H. pylori infection (3h). NRF2 protein stability was reduced following FGFR4 or P62 knockdown. The half-life time (t1/2) of NRF2 was calculated using GraphPad Prism software and plotted on the right side of panels C and D, respectively. *P < 0.05, **P < 0.001.

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Western Blot, Infection, Knockdown, Software

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 7. FGFR4, P62, and KEAP1 coexist in the same protein complex. A-B) Immunoprecipitation and western blot analysis following P62 pulldown (A) or FGFR4 pulldown (B) using MKN28 cells infected with H. pylori 7.13 (3 h). Immunoprecipitations and their corresponding input samples were subjected to immunoblotting with P62 FGFR4 Keap1 and NRF2 antibodies. The infection was confirmed using CagA antibody and equal amounts of protein loading were confirmed in the input samples using GAPDH antibody. C) Proximity ligation assay was performed in MKN28 cells transfected with control or FGFR4 siRNA and infected with H. pylori (7.13). The presence of red signals indicates positive ligation and proximity of the proteins, indicative of interaction. Using FGFR4 and P62 antibodies, the results indicated the presence of FGFR4-P62 interaction (red signals) following H. pylori infection (left upper panel). This interaction was not detected with FGFR4 siRNA (middle upper panel). The lower panels display the negative control for PLA background reaction. Control cells were transfected with Ctrl siRNA, infected with H. pylori and probed with a single antibody against FGFR4 (lower left panel) or P62 (lower right panel). The upper right panel displays a negative control for the PLA background with no antibody. Maximum intensity projection is presented on the right and lower sides of each image. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Immunoprecipitation, Western Blot, Infection, Proximity Ligation Assay, Transfection, Control, Ligation, Negative Control

Journal: Redox biology

Article Title: Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis.

doi: 10.1016/j.redox.2023.102998

Figure Lengend Snippet: Fig. 9. A schematic diagram illustrating the role of FGFR4 in activating and stabilizing NRF2. Exposure of cells to H. pylori infection induces oxidative stress and FGFR4 expression. FGFR4 binds to P62. The FGFR4-P62 complex binds to KEAP1 in NRF2 degradation complex, competing with binding of KEAP1 top NRF2. NRF2 escapes degradation, accumulates, and translocates to the nucleus to induce transcription of antioxidant response genes.

Article Snippet: For NRF2 and FGFR4 detection, 100ul of (1:200) rabbit NRF2 antibody (Novus Biologicals), 1:200 mouse FGFR4 antibody (Santa Cruz Biotechnology), were added for overnight in the dark.

Techniques: Infection, Expressing, Binding Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models.

doi: 10.1186/s13046-023-02838-3

Figure Lengend Snippet: Fig. 2 FGFR4 expression in RMS cell lines and PDXs. A FGFR4 expression levels were evaluated in eleven RMS cell lines, three PDXs, and in myoblasts, PBMCs, and T cells as controls. WB results revealed high expression in Rh36, Rh4, Rh28, JR, and RMS cell lines, whereas low levels were detected in Rh18, RUCH-3, Rh5, and Rh30. No FGFR4 expression was observed in the controls. B Flow Cytometry results confirmed expression of FGFR4 on the surface of most RMS cell lines, especially high in Rh4, Rh18, and JR. The corresponding isotype controls are highlighted in grey. C Estimation of FGFR4 expression on RMS cell lines by Quantibrite PE-Beads showed a surface expression in the range of 6’00–4’000 copies per cell

Article Snippet: Immunohistochemistry primary antibodies and working dilutions: mouse monoclonal anti-human FGFR4 (1:50; Santa Cruz, #SC-136988), goat polyclonal anti-CD276 (1 μg/ml; R&D systems, #AF1027), rabbit monoclonal anti-CD3 (1:200; Abcam, #16669).

Techniques: Expressing, Flow Cytometry

Journal: Journal of experimental & clinical cancer research : CR

Article Title: CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models.

doi: 10.1186/s13046-023-02838-3

Figure Lengend Snippet: Fig. 4 Schematic representation of the investigated CAR constructs. A The backbone used for the expression of the CARs is characterized by an EFS promoter, followed by a Kozak sequence and a CD28 leader sequence. The ORF is composed of two MycTag regions; an Antigen Binding Domain (ABD; anti-CD19 scFv FMC63 for the initial cloning) flanked by two Esp3I restriction sites for ABD swapping with anti-CD276 scFv (CD276.MG) or with FGFR4-targeting F8-FR4 sdAbs; a Hinge Domain (HD) and a Transmembrane domain (TM) derived from either CD8α or CD28; one or two Co-Stimulatory Domains (CSDs), derived from CD28 or 4-1BB; and a CD3ζ Signaling Domain (SD). Downstream, GFP as reporter is linked to the CD3ζ domain by the self-cleaving peptide P2A. B Modular CAR structure of the different constructs generated by Gibson assembly with either CD8α HD/TM (green) or CD28 HD/TM (cyan). In each panel, the schematic representation of two second-generation CARs, characterized by a single CSD (CD28 or 4-1BB), and two third-generation CARs, characterized by the expression of both CSDs (CD28-4-1BB or 4-1BB-CD28). Created with Biorender. com

Article Snippet: Immunohistochemistry primary antibodies and working dilutions: mouse monoclonal anti-human FGFR4 (1:50; Santa Cruz, #SC-136988), goat polyclonal anti-CD276 (1 μg/ml; R&D systems, #AF1027), rabbit monoclonal anti-CD3 (1:200; Abcam, #16669).

Techniques: Construct, Expressing, Sequencing, Binding Assay, Cloning, Derivative Assay, Generated

Journal: Journal of experimental & clinical cancer research : CR

Article Title: CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models.

doi: 10.1186/s13046-023-02838-3

Figure Lengend Snippet: Fig. 7 Evaluation of killing capacity by CD276- and F8-FR4-CAR T cells after co-incubation with fLuc+ RD, Rh4, and JR cell lines. In order to determine the CAR construct conferring the highest cytotoxicity against RMS, CD276-CAR T cells were co-incubated for 48 h with fLuc+ cells RD (A) and Rh4 (B) at different E:T ratios. F8-FR4-CAR T cells were co-incubated with fLuc+ JR (C) and Rh4 (D) since RD cells express very low amounts of FGFR4. Luciferase assays showed significant killing capacity by all the generations of CAR T cells at E:T ratios of 5:1 and 10:1, compared to untransduced T cells (UTD) (p < 0.0001 for all constructs). Lower E:T ratios highlighted optimal efficacy by CD276.V- and F8-FR4.V-CAR T cells, compared to the other constructs. CD19-CAR T cells showed no cytotoxic effect when co-incubated with fLuc+ RD (E) and Rh4 (F) cells. As positive controls, CD19.V-CAR T cells were incubated with fLuc+ RD-tCD19 (E, grey) and Rh4-tCD19 (F, grey) cells. All experiments were performed in triplicates and with three different donors. Donor-specific results and detailed statistical analyses are described in Suppl. Fig. S4 and Suppl. Table S9, respectively. The statistical significance of the differences between experimental and control groups was assessed using Dunnett’s multiple comparison test following a two-way ANOVA; p ≤ 0.01 (**), p ≤ 0.0001 (****)

Article Snippet: Immunohistochemistry primary antibodies and working dilutions: mouse monoclonal anti-human FGFR4 (1:50; Santa Cruz, #SC-136988), goat polyclonal anti-CD276 (1 μg/ml; R&D systems, #AF1027), rabbit monoclonal anti-CD3 (1:200; Abcam, #16669).

Techniques: Incubation, Construct, Luciferase, Control, Comparison

Journal: Journal of experimental & clinical cancer research : CR

Article Title: CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models.

doi: 10.1186/s13046-023-02838-3

Figure Lengend Snippet: Fig. 15 Expression of CD276 and FGFR4 on RMS-derived xenografts. A Expression of CD276 was assessed by Flow Cytometry on untreated Rh4-derived xenograft tumors and compared to cultured wild-type Rh4 cells by using a PE-conjugated anti-CD276 antibody. CD276 expression was calculated dividing the geometric mean of antibody-stained cells by the geometric mean of the controls. CD276 expression showed a lower CD276 expression on Rh4 tumor-derived cells, compared to the cultured Rh4 cells. B Expression of FGFR4 was assessed by Flow Cytometry on Rh4-derived xenograft tumors from untreated mice and compared to cultured wild-type Rh4 cells by using a PE-conjugated anti-FGFR4 antibody. FGFR4 expression was calculated dividing the geometric mean of antibody-stained cells by the geometric mean of the isotype controls. The results indicated a strong decrease in FGFR4 expression in Rh4 tumor xenograft-derived cells. C Assessment of CD276 and FGFR4 expression in tumor xenografts tissue by IHC. After treatment, CD276 is still highly detectable on RD- and Rh4-derived tumor xenografts, but low staining detection was observed in JR-derived tumors. FGFR4 seems expressed at low levels on RD, at medium levels on JR, and at high levels on Rh4

Article Snippet: Immunohistochemistry primary antibodies and working dilutions: mouse monoclonal anti-human FGFR4 (1:50; Santa Cruz, #SC-136988), goat polyclonal anti-CD276 (1 μg/ml; R&D systems, #AF1027), rabbit monoclonal anti-CD3 (1:200; Abcam, #16669).

Techniques: Expressing, Derivative Assay, Flow Cytometry, Cell Culture, Staining

Journal: Journal of experimental & clinical cancer research : CR

Article Title: CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models.

doi: 10.1186/s13046-023-02838-3

Figure Lengend Snippet: Fig. 2 FGFR4 expression in RMS cell lines and PDXs. A FGFR4 expression levels were evaluated in eleven RMS cell lines, three PDXs, and in myoblasts, PBMCs, and T cells as controls. WB results revealed high expression in Rh36, Rh4, Rh28, JR, and RMS cell lines, whereas low levels were detected in Rh18, RUCH-3, Rh5, and Rh30. No FGFR4 expression was observed in the controls. B Flow Cytometry results confirmed expression of FGFR4 on the surface of most RMS cell lines, especially high in Rh4, Rh18, and JR. The corresponding isotype controls are highlighted in grey. C Estimation of FGFR4 expression on RMS cell lines by Quantibrite PE-Beads showed a surface expression in the range of 6’00–4’000 copies per cell

Article Snippet: Western Blotting primary antibodies and working dilutions used were: goat anti-CD276 (1 μg/ml; R&D systems, #AF1027); mouse anti-α-tubulin (1:1′000; Cell Signaling, #2144); rabbit anti-MycTag (1:1′000; Cell Signaling, #2278S); mouse anti-human FGFR4 (Santa Cruz, #SC-136988).

Techniques: Expressing, Flow Cytometry

Journal: Journal of experimental & clinical cancer research : CR

Article Title: CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models.

doi: 10.1186/s13046-023-02838-3

Figure Lengend Snippet: Fig. 4 Schematic representation of the investigated CAR constructs. A The backbone used for the expression of the CARs is characterized by an EFS promoter, followed by a Kozak sequence and a CD28 leader sequence. The ORF is composed of two MycTag regions; an Antigen Binding Domain (ABD; anti-CD19 scFv FMC63 for the initial cloning) flanked by two Esp3I restriction sites for ABD swapping with anti-CD276 scFv (CD276.MG) or with FGFR4-targeting F8-FR4 sdAbs; a Hinge Domain (HD) and a Transmembrane domain (TM) derived from either CD8α or CD28; one or two Co-Stimulatory Domains (CSDs), derived from CD28 or 4-1BB; and a CD3ζ Signaling Domain (SD). Downstream, GFP as reporter is linked to the CD3ζ domain by the self-cleaving peptide P2A. B Modular CAR structure of the different constructs generated by Gibson assembly with either CD8α HD/TM (green) or CD28 HD/TM (cyan). In each panel, the schematic representation of two second-generation CARs, characterized by a single CSD (CD28 or 4-1BB), and two third-generation CARs, characterized by the expression of both CSDs (CD28-4-1BB or 4-1BB-CD28). Created with Biorender. com

Article Snippet: Western Blotting primary antibodies and working dilutions used were: goat anti-CD276 (1 μg/ml; R&D systems, #AF1027); mouse anti-α-tubulin (1:1′000; Cell Signaling, #2144); rabbit anti-MycTag (1:1′000; Cell Signaling, #2278S); mouse anti-human FGFR4 (Santa Cruz, #SC-136988).

Techniques: Construct, Expressing, Sequencing, Binding Assay, Cloning, Derivative Assay, Generated

Journal: Journal of experimental & clinical cancer research : CR

Article Title: CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models.

doi: 10.1186/s13046-023-02838-3

Figure Lengend Snippet: Fig. 7 Evaluation of killing capacity by CD276- and F8-FR4-CAR T cells after co-incubation with fLuc+ RD, Rh4, and JR cell lines. In order to determine the CAR construct conferring the highest cytotoxicity against RMS, CD276-CAR T cells were co-incubated for 48 h with fLuc+ cells RD (A) and Rh4 (B) at different E:T ratios. F8-FR4-CAR T cells were co-incubated with fLuc+ JR (C) and Rh4 (D) since RD cells express very low amounts of FGFR4. Luciferase assays showed significant killing capacity by all the generations of CAR T cells at E:T ratios of 5:1 and 10:1, compared to untransduced T cells (UTD) (p < 0.0001 for all constructs). Lower E:T ratios highlighted optimal efficacy by CD276.V- and F8-FR4.V-CAR T cells, compared to the other constructs. CD19-CAR T cells showed no cytotoxic effect when co-incubated with fLuc+ RD (E) and Rh4 (F) cells. As positive controls, CD19.V-CAR T cells were incubated with fLuc+ RD-tCD19 (E, grey) and Rh4-tCD19 (F, grey) cells. All experiments were performed in triplicates and with three different donors. Donor-specific results and detailed statistical analyses are described in Suppl. Fig. S4 and Suppl. Table S9, respectively. The statistical significance of the differences between experimental and control groups was assessed using Dunnett’s multiple comparison test following a two-way ANOVA; p ≤ 0.01 (**), p ≤ 0.0001 (****)

Article Snippet: Western Blotting primary antibodies and working dilutions used were: goat anti-CD276 (1 μg/ml; R&D systems, #AF1027); mouse anti-α-tubulin (1:1′000; Cell Signaling, #2144); rabbit anti-MycTag (1:1′000; Cell Signaling, #2278S); mouse anti-human FGFR4 (Santa Cruz, #SC-136988).

Techniques: Incubation, Construct, Luciferase, Control, Comparison

Journal: Journal of experimental & clinical cancer research : CR

Article Title: CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models.

doi: 10.1186/s13046-023-02838-3

Figure Lengend Snippet: Fig. 15 Expression of CD276 and FGFR4 on RMS-derived xenografts. A Expression of CD276 was assessed by Flow Cytometry on untreated Rh4-derived xenograft tumors and compared to cultured wild-type Rh4 cells by using a PE-conjugated anti-CD276 antibody. CD276 expression was calculated dividing the geometric mean of antibody-stained cells by the geometric mean of the controls. CD276 expression showed a lower CD276 expression on Rh4 tumor-derived cells, compared to the cultured Rh4 cells. B Expression of FGFR4 was assessed by Flow Cytometry on Rh4-derived xenograft tumors from untreated mice and compared to cultured wild-type Rh4 cells by using a PE-conjugated anti-FGFR4 antibody. FGFR4 expression was calculated dividing the geometric mean of antibody-stained cells by the geometric mean of the isotype controls. The results indicated a strong decrease in FGFR4 expression in Rh4 tumor xenograft-derived cells. C Assessment of CD276 and FGFR4 expression in tumor xenografts tissue by IHC. After treatment, CD276 is still highly detectable on RD- and Rh4-derived tumor xenografts, but low staining detection was observed in JR-derived tumors. FGFR4 seems expressed at low levels on RD, at medium levels on JR, and at high levels on Rh4

Article Snippet: Western Blotting primary antibodies and working dilutions used were: goat anti-CD276 (1 μg/ml; R&D systems, #AF1027); mouse anti-α-tubulin (1:1′000; Cell Signaling, #2144); rabbit anti-MycTag (1:1′000; Cell Signaling, #2278S); mouse anti-human FGFR4 (Santa Cruz, #SC-136988).

Techniques: Expressing, Derivative Assay, Flow Cytometry, Cell Culture, Staining