Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 suppresses MST1/2 activation and nuclear localization in cancer cell spheres. a shScr and shFGFR4 MDA-MB-453 cell spheres were cultured under non-adherent conditions (10% or 2% FBS), and subjected to immunoblotting. Arrowhead; cleaved N-terminal MST1/2 (in 2% FBS), brackets highlight the fragments of autoactivated MST1/2. b MDA-MB-453 cell spheres were treated with 100 n m BLU9931 for 15 min, and subjected to immunoblotting. c , d shScr and shFGFR4 MDA-MB-453 and ZR-75.1 spheres were analyzed for MST1 expression by c immunofluorescence, and d MST1 nuclear/cytoplasmic ratio was quantified ( n = 4–6 MDA-MB-453 spheres, ≥ 6 microscopic fields/sphere; n = 2–3 ZR-75.1 spheres, ≥ 8 microscopic fields/ sphere; mean ± SEM of two independent experiments. Scale bar 10 µm. e shScr and shFGFR4 MDA-MB-453 cells were transfected with indicated siRNAs before sphere formation, cultured under non-adherent conditions (1% FBS) for 48 h, and subjected to immunoblotting

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Activation Assay, Cell Culture, Western Blot, Expressing, Immunofluorescence, Transfection

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: MST1-Y433F phosphosite mutant restores MST1/2 activation in FGFR4 expressing cancer cells. a MDA-MB-231 cells co-transfected with FGFR4 (R) and wild-type or phosphosite mutant MST1-Y433F were subjected to immunoblotting as indicated. Ratio of pMOB1/MOB1 is indicated below the immunoblot panel. b T47D cells (co-)transfected with wild-type or MST1-Y433F alone or with FGFR4 (R) were treated with 1 µ m okadaic acid for 1 h before cell lysis, and subjected to immunoblotting. See corresponding T47D immunoblots without okadaic acid in Fig. S4C. c T47D cells with indicated siRNAs, and (co-)transfected with wild-type or MST1-Y433F alone or with FGFR4 (R) were treated with 1 µ m okadaic acid as above, and subjected to immunoblotting. a–c Brackets and arrowhead indicate the activated pMST1/2 fragments. N = 2 independent repeats

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Phospho-proteomics, Mutagenesis, Activation Assay, Expressing, Transfection, Western Blot, Lysis

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 substrate screen identifies tyrosine-phosphorylated Hippo pathway proteins including MST1/2. a Scheme of the substrate screen with recombinant FGFR4 kinase domain. b Top 10 FGFR4 substrates ranked by the Z-score include Hippo pathway -associated proteins (yellow). See Table S1 for the full substrate list. c , d MST1/2 are tyrosine phosphorylated by FGFR4 in COS-1 cells. Flag-tagged MST1/2 were immunoprecipitated after transfection of MST1 and MST2 alone or in combination with FGFR4 G388 (G), or R388 (R) kinase (wt), or kinase-dead (KD) variants, and detected by immunoblotting. e MST1 immunoprecipitates from COS-1 cells co-transfected with FGFR4 (R)-wt or FGFR4 (R)-KD (See Fig. S1A) were trypsin digested and subjected to phoshopeptide enrichment prior to LC-MS/MS analysis ( N = 3) that identified phosphorylated Y433 (red) on MST1 only with FGFR4 (R)-wt, and phosphorylated S410 (green) only with FGFR4 (R)-KD

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Recombinant, Immunoprecipitation, Transfection, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: List of MST1 phoshopeptides identified by mass spectrometry

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Sequencing

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 is overexpressed in HER2 + , MST1/2 low breast cancer cells. a , b FGFR4 and HER2 expression in luminal MDA-MB-453, ZR-75.1, and BT474, MCF7, and T47D, and five triple-negative breast cancer cell lines by a immunoblotting and b immunofluorescence. Scale bar 20 μm. c MST1, MST2, and YAP/TAZ expression in these cell lines, detected by immunoblotting ( N = 3)

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 suppresses MST1/2 activation and cleavage in HER2 + breast cancer cells. a , b MDA-MB-453 cells transfected with indicated siRNAs were subjected to immunoblotting for a T183/180 phosphorylated MST1/2, and b MST1 and MST2. Note cleaved ~ 37 kDa MST1/N in FGFR4 knockdown cells (arrowhead). Thin gray line indicates cropping to leave out irrelevant sample lane; see uncropped immunoblots in Fig. S8. c MDA-MB-453 cells transduced with indicated shRNAs were transfected with siScr or siFGFR4 siRNA to 3’UTR before transfection of mock or FGFR4 (R) or (G) overexpression plasmid for a rescue experiment. Lysates were subjected to immunoblotting as indicated. Brackets indicate the cleaved MST1 and MST2 fragments. See Fig. S2A for phopsho-FRS2α and short exposure of MST1. d MDA-MB-453 and ZR-75.1 cells were transduced with indicated si/shRNAs; upper, indicated immunoblots of lysates; lower, quantification of pMOB1/MOB1 ratio, N = 3, mean ± SEM; * P < 0.05. For MST1/2 knockdown e ZR-75.1 and f MDA-MB-453 were transduced with shRNAs followed by transfection with siRNAs as indicated, and g BT474 cells were transfected with indicated siRNAs, and subjected to immunoblotting for pT183/180 MST1/2, MST1, MST2, and pMOB1 as indicated (in e arrowhead points to a full-length, bracket to the cleaved MST2) a–g . N = 3 independent repeats for all; except N = 2 in f and g

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Activation Assay, Transfection, Western Blot, Knockdown, Transduction, Over Expression, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 counteracts MST1/2-mediated apoptosis. MDA-MB-453 cells transduced with shScr or shFGFR4 shRNAs were transfected with siRNA pools specific for FGFR4, MST1 or MST2, and analyzed for annexin V and propidium iodide (PI) binding by flow cytometry using two different gating strategies for data visualization. a Gating to populations P1 (smaller) and P2 (larger), and annexin V binding (FL1-A) histograms as a marker for early apoptotic cells. b Quantification (% of total, 100,000 events) of apoptosis based on double-positive (annexin V + PI) cells, including both early and late apoptotic stages. See Fig. S3B for representative contour plots and quadrant gating. Mean ± SD of triplicates shown, ** P < 0.01; (repeated three times; N = 3). FSC-A; forward scatter, and SSC-A; side scatter

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Transduction, Transfection, Binding Assay, Flow Cytometry, Marker

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 confers resistance to apoptotic modulators in comprehensive drug screen. a (Phospho)protein changes in TCGA RPPA data associated with FGFR4 upregulation in breast cancer, visualized using cBioPortal (RPPA score change in breast cancer tumors with and without alterations in FGFR4; (mean FGFR4 altered – mean FGFR4 unaltered) [ , ]. The most significantly up- and downregulated proteins are highlighted (pink dots); ERBB2, alternative name of HER2; PR, progesterone receptor. b–g Fibrin embedded single-cell suspensions of b–d MDA-MB-453 and e–g ZR-75.1 cells were treated with 100 n m BLU9931 and/or 30 ng/ml FGF1 over a 13–14-day culture, fixed, embedded into paraffin for sectioning, and subjected to immunohistochemistry for Ki67 and BAX expression. Positively stained vs. total number of cells per colony were counted ( N = 30, mean ± SD, ** P < 0.01). Scale bar 50 µm in b and e . b For comprehensive drug sensitivity testing ( N = 1), MDA-MB-453 cells were treated with 527 compounds in five-point dose either alone or in combination with specific FGFR4 inhibitor BLU9931. Dotplot showing the difference in DSS (drug sensitivity score) for cells in treatment combination with BLU9931 (100 n m ) versus single agent treatments. Negative values are compounds inducing larger decreases in viability as single agents; positive scores indicate compounds yielding larger decreases in viability in the presence of BLU9931. Colors demarcate compounds with similar class

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Immunohistochemistry, Expressing, Staining

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: After washing, sections were incubated overnight at 4 ◦C with the primary antibody against FGFR4 (1:1000) ProteinTech, NRF2 (1:1000) Abcam or P62 (1:2000) Cell Signaling.

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: After washing, sections were incubated overnight at 4 ◦C with the primary antibody against FGFR4 (1:1000) ProteinTech, NRF2 (1:1000) Abcam or P62 (1:2000) Cell Signaling.

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: After washing, sections were incubated overnight at 4 ◦C with the primary antibody against FGFR4 (1:1000) ProteinTech, NRF2 (1:1000) Abcam or P62 (1:2000) Cell Signaling.

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: After washing, sections were incubated overnight at 4 ◦C with the primary antibody against FGFR4 (1:1000) ProteinTech, NRF2 (1:1000) Abcam or P62 (1:2000) Cell Signaling.

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: After washing, sections were incubated overnight at 4 ◦C with the primary antibody against FGFR4 (1:1000) ProteinTech, NRF2 (1:1000) Abcam or P62 (1:2000) Cell Signaling.

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: After washing, sections were incubated overnight at 4 ◦C with the primary antibody against FGFR4 (1:1000) ProteinTech, NRF2 (1:1000) Abcam or P62 (1:2000) Cell Signaling.

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: After washing, sections were incubated overnight at 4 ◦C with the primary antibody against FGFR4 (1:1000) ProteinTech, NRF2 (1:1000) Abcam or P62 (1:2000) Cell Signaling.

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: After washing, sections were incubated overnight at 4 ◦C with the primary antibody against FGFR4 (1:1000) ProteinTech, NRF2 (1:1000) Abcam or P62 (1:2000) Cell Signaling.

Techniques: Infection, Expressing, Binding Assay

Journal: Oncotarget

Article Title: Up-regulation of fibroblast growth factor 19 and its receptor associates with progression from fatty liver to hepatocellular carcinoma

doi: 10.18632/oncotarget.10750

Figure Lengend Snippet: ( A ) the protein levels of FGF19 in serum from 24 HCC patients and 6 non-HCC controls, and in the tissues from 24 paired HCC-peritumoral and 6 non-HCC controls by ELISA analysis. ( B ) the mRNA levels of FGF19 and FGFR4 from 24 paired HCC-peritumoral tissues. ( C ) upper: Representative Western blot for β-Klotho protein detection of 5 paired tissues from HCC patients. Lower: the protein levels of β-Klotho in serum from 24 HCC patients and 6 non-HCC controls, and in the tissues from 24 paired HCC-peritumoral tissues by ELISA analysis. T: HCC tumor tissue; PT: peritumoral tissues.

Article Snippet: After rewashing, the slides were incubated separately with the monoclonal mouse FGF19 antibody (1:100), FGFR4 antibody (1:100), or EpCAM antibody (1:100) (SantaCruz Biotechnology Inc, CA) for 30 minutes at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Oncotarget

Article Title: Up-regulation of fibroblast growth factor 19 and its receptor associates with progression from fatty liver to hepatocellular carcinoma

doi: 10.18632/oncotarget.10750

Figure Lengend Snippet: Representative immunohistochemical staining for FGFR4 and the computer quantification of FGFR4 expression from different stages. Magnification: 200×, the positive staining shown as brown color. PT: peritumoral tissues; ST: steatosis with diffuse lipid deposition; NASH: lipid deposition with inflammatory cells infiltration; CR: cirrhosis with regenerative nodule; HCC: hepatocellular carcinoma. * p < 0.05 vs PT.

Article Snippet: After rewashing, the slides were incubated separately with the monoclonal mouse FGF19 antibody (1:100), FGFR4 antibody (1:100), or EpCAM antibody (1:100) (SantaCruz Biotechnology Inc, CA) for 30 minutes at room temperature.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Oncotarget

Article Title: Up-regulation of fibroblast growth factor 19 and its receptor associates with progression from fatty liver to hepatocellular carcinoma

doi: 10.18632/oncotarget.10750

Figure Lengend Snippet: ( A ) Positive correlation of FGF19 and FGFR4 expression in HCC ( r = 0.79) (p < 0.001). ( B ) Positive correlation of FGF19 and EpCAM expression in HCC ( r = 0.852) ( p < 0.001).

Article Snippet: After rewashing, the slides were incubated separately with the monoclonal mouse FGF19 antibody (1:100), FGFR4 antibody (1:100), or EpCAM antibody (1:100) (SantaCruz Biotechnology Inc, CA) for 30 minutes at room temperature.

Techniques: Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Goblet Cell Differentiation Potential in Human Corneal Limbal Epithelial Progenitor Cells In Vitro

doi: 10.1167/iovs.61.12.27

Figure Lengend Snippet: FGFR, Fibroblast Growth Factor Receptor

Article Snippet: The following Abs were used: mouse anti-cytokeratin-3/2p monoclonal antibody (mAb; AE-5, 1:100; Santa Cruz Biotechnology, Inc, Dallas, TX, USA), mouse anti-human cytokeratin-4 mAb (6B10, 1:300; Abcam, Cambridge, UK), rabbit monoclonal [EP1599Y] anti-human cytokeratin 4 (1:200, Abcam), mouse anti-human cytokeratin-7 mAb (RCK105, 1:10000; Millipore, Billerica, MA, USA), mouse anti-human cytokeratin-13 mAb (1:20; American Research Products, Inc., Palos Verdes, CA, USA), mouse anti-human cytokeratin-12 mAb (N-16; 1:500; Santa Cruz Biotechnology), rabbit anti-cytokeratin 12/K12 mAb (1:200, EPR17882, Abcam), rabbit anti-human MUC5AC polyclonal Ab (H-160, 1:100, Santa Cruz Biotechnology), anti-FGFR1 mAb (1:200, 133105; R&D Systems, Minneapolis, MN, USA), anti-human FGFR2 (blocking) mAb (1:200, 98725; R&D Systems), anti-FGFR3 mAb (1:200, 135334; R&D Systems), anti-FGFR4 mAb (1:200, 137114; R&D Systems), and anti-FGFR1 blocking mAb (VBS1; Chemicon, Temecula, CA, USA).

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Goblet Cell Differentiation Potential in Human Corneal Limbal Epithelial Progenitor Cells In Vitro

doi: 10.1167/iovs.61.12.27

Figure Lengend Snippet: FGF receptor expression and effect of FGF receptor blockade in colonies derived from adherent single cells. ( A ) FGFR1 and FGFR2, but not FGFR3 and FGFR4, are detected in the adherent colonies by RT-PCR. ( B ) FGFR1 and FGFR2 expression is detected by Western blotting. ( C ) Effect of FGF receptor blockade was tested in colonies derived from adherent single cells. PAS-positive colony number in the anti-FGFR1 monoclonal antibody group significantly decreases as compared with that in the control Ig group. Treatment with an anti-FGFR2 blocking mAb does not affect the number of colony. ( D ) Colony with amorphous material and epithelium is observed with control IgG. ( E ) Anti-FGFR1 mAb suppresses the proliferation of both goblet-like cell and epithelium. Similar results were obtained with repeated three experiments. M, size markers; S, sample; P, positive control; N, negative control, N.S., not significant.

Article Snippet: The following Abs were used: mouse anti-cytokeratin-3/2p monoclonal antibody (mAb; AE-5, 1:100; Santa Cruz Biotechnology, Inc, Dallas, TX, USA), mouse anti-human cytokeratin-4 mAb (6B10, 1:300; Abcam, Cambridge, UK), rabbit monoclonal [EP1599Y] anti-human cytokeratin 4 (1:200, Abcam), mouse anti-human cytokeratin-7 mAb (RCK105, 1:10000; Millipore, Billerica, MA, USA), mouse anti-human cytokeratin-13 mAb (1:20; American Research Products, Inc., Palos Verdes, CA, USA), mouse anti-human cytokeratin-12 mAb (N-16; 1:500; Santa Cruz Biotechnology), rabbit anti-cytokeratin 12/K12 mAb (1:200, EPR17882, Abcam), rabbit anti-human MUC5AC polyclonal Ab (H-160, 1:100, Santa Cruz Biotechnology), anti-FGFR1 mAb (1:200, 133105; R&D Systems, Minneapolis, MN, USA), anti-human FGFR2 (blocking) mAb (1:200, 98725; R&D Systems), anti-FGFR3 mAb (1:200, 135334; R&D Systems), anti-FGFR4 mAb (1:200, 137114; R&D Systems), and anti-FGFR1 blocking mAb (VBS1; Chemicon, Temecula, CA, USA).

Techniques: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Blocking Assay, Positive Control, Negative Control

Journal: Frontiers in Oncology

Article Title: FGF19 Is Coamplified With CCND1 to Promote Proliferation in Lung Squamous Cell Carcinoma and Their Combined Inhibition Shows Improved Efficacy

doi: 10.3389/fonc.2022.846744

Figure Lengend Snippet: FGF19 enhances CCND1 expression by FGF19-FGFR4-ERK1/2 axis in LUSC cells. (A) Recombinant human FGF19 (rhFGF19) (25 ng/ml) promoted expression of CCND1 in SK-MES-1 and H520 cells (serum starved for 12 h before treatment) in a time-dependent manner. (B) CCND1 mRNA expression levels in LUSC cell lines after treatment with rhFGF2 for 12 h. (C) Expression of CCND1 in FGF19 overexpression LUSC cell line. (D) Expression of CCND1 in FGF19 knockdown LUSC cell line. (E) A panel of inhibitors against a number of signaling pathways was used to dissect the leading factors of regulated by FGF19. (F) Western blot and (G) qPCR analysis of CCND1 expression in H520 and HCC95 LUSC cells after treatment of FGF19, FGF19 and SCH772984, FGF19 & BLU9931, or DMSO as control. (H) Effects of the FGF19/FGFR4 pathway on protein levels of CCND1, p-FGFR4, and p-ERK1/2 by western blot analysis. H520, SK-MES-1 and HCC95 cells were treated with FGF19 (25 ng/ml, 0/0.5/6/12 h) to activate the FGF19/FGFR4 signaling pathway. Data were shown as mean ± SD bars and compared by unpaired t -test. ** p < 0.01; **** p < 0.0001; ns, not significant.

Article Snippet: Antibodies for western blot were as follows: β-Actin (Proteintech,66009-1-Ig), β-Tubulin ( Proteintech, 66240-1-Ig ) , FGF19 (R&D System, AF969), FGFR4 (R&D System, MAB6852), FRS2 (Abcam, ab10425), pFRS2 (R&D System, AF5126), goat anti-mouse IgG-HRP (Jackson ImmunoResearch, 115-035-003), goat anti-rabbit IgG-HRP (Jackson ImmunoResearch, 111-035-003), donkey anti-Goat IgG-HRP (Sangon Biotech, D110115) and other antibodies were obtained from the Cell Signaling Technology.

Techniques: Expressing, Recombinant, Over Expression, Knockdown, Protein-Protein interactions, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: FGF19 Is Coamplified With CCND1 to Promote Proliferation in Lung Squamous Cell Carcinoma and Their Combined Inhibition Shows Improved Efficacy

doi: 10.3389/fonc.2022.846744

Figure Lengend Snippet: The schematic model of this study: underlying mechanism that link neighboring oncogene FGF19 & CCND1 co-amplification in the development of LUSC. FGF19 was co-amplified and co-expressed with its neighboring gene CCND1 in a subset of LUSC. Overexpression of FGF19 activates FGFR4 and leads to phosphorylation of FRS2 and ERK1/2, which further strengthens the increase in CCND1 expression caused by amplification. These lead to the phosphorylation of RB by CCND1-CDK4/6 complex, causing unrestrained transcription factor E2F, which promotes cell cycle progression and lead to the malignant proliferation of LUSC. While co-targeting FGFR4 and CDK4/6 by BLU9931 and palbociclib potentiated the growth inhibition and arrested cells in G1 phase.

Article Snippet: Antibodies for western blot were as follows: β-Actin (Proteintech,66009-1-Ig), β-Tubulin ( Proteintech, 66240-1-Ig ) , FGF19 (R&D System, AF969), FGFR4 (R&D System, MAB6852), FRS2 (Abcam, ab10425), pFRS2 (R&D System, AF5126), goat anti-mouse IgG-HRP (Jackson ImmunoResearch, 115-035-003), goat anti-rabbit IgG-HRP (Jackson ImmunoResearch, 111-035-003), donkey anti-Goat IgG-HRP (Sangon Biotech, D110115) and other antibodies were obtained from the Cell Signaling Technology.

Techniques: Amplification, Over Expression, Phospho-proteomics, Expressing, Inhibition

Journal: Gut

Article Title: Control of hepatocyte proliferation and survival by Fgf receptors is essential for liver regeneration in mice.

doi: 10.1136/gutjnl-2014-307874

Figure Lengend Snippet: Figure 1 Impaired liver regeneration in mice after knockdown of Fgfr4 in hepatocytes. (A) Schematic representation of the experimental setup. Mice were injected twice with Fgfr4 or Luc siRNA with a time interval of 5 days. After additional 5 days, they were subjected to two-third (partial) hepatectomy (PH). (B) Knockdown of Fgfr4 was verified by qRT-PCR using RNA from total liver or cultured primary hepatocytes. N=3–7 per treatment group. Expression levels in mice treated with Luc siRNA were arbitrarily set as 1. (C) Activities of ALT and AST in the serum were determined in non-injured mice (n.i.) and at different time points after PH (N=3–9). (D) Morphometric analysis of the necrotic damage using H&E-stained liver sections at different time points after PH in mice injected with Fgfr4 siRNA and at 48 h in mice injected with Fgfr4-2 siRNA. N=4–9. Representative sections from mice 48 h after PH are shown below. Necrotic areas are encircled with dotted lines. Scale bar: 100 μM. (E) BrdU-positive cells were counted in liver sections at different time points after PH in mice injected with Fgfr4 siRNA and at 48 h in mice injected with Fgfr4-2 siRNA. N=4–9. Representative sections stained with a BrdU antibody are shown for the 48 h time point. Scale bar: 50 μM. (F) Levels of Fgf15 in the serum were determined in non-injected mice and at different time points after PH. N=3–5. All bars represent mean ±SEM. ALT, alanine transaminase; AST, aspartate transaminase; BrdU, 5-bromo-20-deoxyuridine; Fgfr, fibroblast growth factor receptor; Luc, luciferase.

Article Snippet: Proteins were analysed by western blotting using antibodies against Fgfr4 (R&D Systems, Minneapolis, Minnesota, USA), Gapdh (HyTest, Turku, Finland), Foxm1 and Cyp7a1 (Santa Cruz, Santa Cruz, California, USA), phospho-STAT3 (Tyr-705) and total-STAT3 (both from Cell Signaling, Beverly, Massachusetts, USA).

Techniques: Knockdown, Injection, Quantitative RT-PCR, Cell Culture, Expressing, Staining, Luciferase

Journal: Gut

Article Title: Control of hepatocyte proliferation and survival by Fgf receptors is essential for liver regeneration in mice.

doi: 10.1136/gutjnl-2014-307874

Figure Lengend Snippet: Figure 2 Compensatory growth of hepatocytes after PH in mice with Fgfr4 knockdown. (A) Liver to body weight ratio in non-injured mice and at different time points after PH in mice injected with Fgfr4 or Luc siRNA. N=3–9 mice per treatment group. (B) Size of hepatocytes as determined on sections stained with rhodamine-coupled phalloidin. (C) The percentage of binucleated cells was determined on sections stained with rhodamine-coupled phalloidin. (D) Cells with mitotic figures (indicated by arrows in the representative stainings) were quantified in H&E-stained sections. Approximately 50 cells from three to seven mice were counted in (B–D). Magnification bar: 50 μm. Bars represent mean±SEM. Fgfr, fibroblast growth factor receptor; Luc, luciferase; PH, partial hepatectomy.

Article Snippet: Proteins were analysed by western blotting using antibodies against Fgfr4 (R&D Systems, Minneapolis, Minnesota, USA), Gapdh (HyTest, Turku, Finland), Foxm1 and Cyp7a1 (Santa Cruz, Santa Cruz, California, USA), phospho-STAT3 (Tyr-705) and total-STAT3 (both from Cell Signaling, Beverly, Massachusetts, USA).

Techniques: Knockdown, Injection, Staining, Luciferase

Journal: Gut

Article Title: Control of hepatocyte proliferation and survival by Fgf receptors is essential for liver regeneration in mice.

doi: 10.1136/gutjnl-2014-307874

Figure Lengend Snippet: Figure 3 Cell cycle progression is impaired in mice with Fgfr4 knockdown. (A–F) qRT-PCR analysis of different cyclins and of Foxm1 and Fxr using RNAs from liver at different time points after PH. N=3–9 per time point and treatment group. Expression levels in non-injured liver of mice injected with Luc siRNA were arbitrarily set as 1. (E and G) Total liver lysates of mice prior to and at different time points after PH were analysed by western blotting for expression of Foxm1 and Gapdh (E), or total and phosphorylated Stat3 (G). The ratio between phosphorylated and total Stat3 is shown. All bars represent mean±SEM. Fgfr, fibroblast growth factor receptor; Luc, luciferase; PH, partial hepatectomy.

Article Snippet: Proteins were analysed by western blotting using antibodies against Fgfr4 (R&D Systems, Minneapolis, Minnesota, USA), Gapdh (HyTest, Turku, Finland), Foxm1 and Cyp7a1 (Santa Cruz, Santa Cruz, California, USA), phospho-STAT3 (Tyr-705) and total-STAT3 (both from Cell Signaling, Beverly, Massachusetts, USA).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Injection, Western Blot, Luciferase

Journal: Gut

Article Title: Control of hepatocyte proliferation and survival by Fgf receptors is essential for liver regeneration in mice.

doi: 10.1136/gutjnl-2014-307874

Figure Lengend Snippet: Figure 4 Fgfr signalling is essential for liver regeneration. Alb-R1/R2 mice or Alb-Cre control mice were injected with Fgfr4 or Luc siRNA and subjected to PH. Survival of mice of all genotypes and treatment groups is shown on (A). The number on top of the bars indicates the number of mice included in the analysis at each time point. (B) Left panel: BrdU-positive cells were counted in liver sections 72 h after PH in Alb-R1/R2 mice and Alb-Cre control mice injected with Fgfr4 or Luc siRNA. N=4–9 per time point and treatment group. Right panels: qRT-PCR analysis of cyclins A2 and B1 using RNAs from liver at 0 h and 72 h after PH. N=3–9 per time point and treatment group. Expression levels in mice injected with Luc siRNA 0 h after PH were arbitrarily set as 1. N=4–9. (C) The necrotic area (encircled with dotted line) was determined in H/E-stained sections. N=4– 9. (D) AST and ALT activities in the serum of mice of all treatment groups at 72 h after PH. N=4–9. (E) Hepatic steatosis as determined by Oil Red O staining of liver sections. Representative sections from 3–10 mice per treatment group are shown. (F) Serum levels of Fgf15 at different time points after PH. N=3–5. (G) Levels of total intrahepatic bile acids were determined using liquid chromatography—mass spectrometry. N=3–5. All bars represent mean±SEM. ALT, alanine transaminase; AST, aspartate transaminase; BrdU, 5-bromo-20-deoxyuridine; Fgfr, fibroblast growth factor receptor; Luc, luciferase; PH, partial hepatectomy.

Article Snippet: Proteins were analysed by western blotting using antibodies against Fgfr4 (R&D Systems, Minneapolis, Minnesota, USA), Gapdh (HyTest, Turku, Finland), Foxm1 and Cyp7a1 (Santa Cruz, Santa Cruz, California, USA), phospho-STAT3 (Tyr-705) and total-STAT3 (both from Cell Signaling, Beverly, Massachusetts, USA).

Techniques: Control, Injection, Quantitative RT-PCR, Expressing, Staining, Liquid Chromatography, Mass Spectrometry, Luciferase