Journal: Journal of cell science

Article Title: STAT1 and STAT3 do not participate in FGF-mediated growth arrest in chondrocytes.

doi: 10.1242/jcs.017160

Figure Lengend Snippet: Fig. 1. FGFR3 interacts with STAT1 and STAT3. FLAG-tagged wt, FGFR3-K650E or FGFR3-K508M were expressed in HeLa cells (A) or RCS chondrocytes (B) and the whole cell lysates were subjected to WB for indicated molecules (left panel). Actin serves as a loading control. The same lysates were subjected to STAT1 (middle panel) and STAT3 (right panel) immunoprecipitation (IP) followed by WB. Please note that the transfected FGFR3- FLAG was detected by FLAG antibody in A and by FGFR3 antibody in B. (C) FLAG- tagged wt or FGFR3-K650E, immunoprecipitated from CHO cells, or recombinant tyrosine kinase (TK) domain of FGFR3 were subjected to an in vitro kinase assay with recombinant STAT1 as a substrate. The level of STAT1 phosphorylation was determined by WB with antibody recognizing STAT1 only when phosphorylated at Y701. Samples including the FGFR inhibitor SU5402 (20 M) or those with ATP omitted serve as negative controls for the kinase reaction. The levels of total STAT1 and FGFR3 serve as controls for the substrate and kinase quantity, respectively. Note that the FGFR3 antibody was raised against the extracellular domain of FGFR3 and thus cannot recognize FGFR3-TK. (D) The kinase assay was carried out as described above, using FGFR3-TK as a kinase, and recombinant STAT1 and/or recombinant FRS2 as substrates. The level of STAT1 phosphorylation was determined as described above, whereas FRS2 tyrosine phosphorylation was determined by WB with the 4G10 phosphotyrosine antibody.

Article Snippet: Antibodies against the following proteins were used: actin, FGFR3 and FRS2 (Santa Cruz Biotechnology, Santa Cruz, CA); ERK1/2, P-ERK1/2T202/Y204, STAT1, STAT3, STAT5, P-STAT1Y701, PSTAT3Y705, P-STAT5Y694, P-STAT1S727, P-STAT3S727 (Cell Signaling, Beverly, MA); FLAG (Sigma, St Louis, MO); SRC (Biosource, Camarillo, CA); and 4G10 (Upstate Biotechnology, Lake Placid, NY).

Techniques: Control, Immunoprecipitation, Transfection, Recombinant, In Vitro, Kinase Assay, Phospho-proteomics

Journal: Human Molecular Genetics

Article Title: Oncogenic FGFR3 gene fusions in bladder cancer

doi: 10.1093/hmg/dds486

Figure Lengend Snippet: ( A ) High-molecular weight forms of FGF receptor 3 (FGFR3) (arrowheads) detected in three non-point mutated bladder tumour cell lines. MGHU3, 94–10 and 97–7 have FGFR3 point mutations. NHUC, normal human urothelial cells. ( B ) Immunoprecipitation with antibodies detecting epitopes in the extracellular (E) or cytoplasmic (C) regions of FGFR3 and detection with FGFR3 antibody B9, which recognizes amino acids 25–124. High-molecular weight forms (arrowheads) are precipitated by E but not C antibody. The FGFR3 Δ8–10 isoform which is expressed at highest level in RT4 is precipitated by C but not E antibody (open arrowhead in RT4-C).

Article Snippet: Sub-confluent TERT-NHUC were removed from flasks by treatment with accutase (Sigma) and allowed to rest in full medium for at least 20 min before staining with monoclonal anti-human FGFR3-phycoerythrin antibody (R&D Systems) according to the manufacturer's instructions.

Techniques: High Molecular Weight, Immunoprecipitation

Journal: Human Molecular Genetics

Article Title: Oncogenic FGFR3 gene fusions in bladder cancer

doi: 10.1093/hmg/dds486

Figure Lengend Snippet: ( A ) FISH to SW780 to define t(4;7) breakpoints. Top row: FISH with bacterial artificial chromosome (BAC) clones from chromosome 4. SW780 has no normal chromosome 4; chromosome 4 which is not involved in t(4;7) is involved in an apparently balanced t(1;4)(q11;q1?) with an apparently intact 4p. RP11-138D6 (green, contains FGFR3 ) hybridizes to intact 4p and both t(4;7) translocation products, while the more distal RP11-572O17 (red) hybridizes to intact 4p and der(7) t(4;7) only. Bottom row: FISH with BACs from chromosome 7. RP11-307C18 (green) hybridizes to normal 7 and both t(4;7) translocation products, while the more distal RP11-473H4 (red) hybridizes to normal 7 and der(4) t(4;7) only. ( B ) RT-PCR products from SW780 cDNA with forward primers from FGFR3 exons 17 or 18 and a reverse primer from BAI1-associated protein 2-like 1 ( BAIAP2L1 ) exon 4. ( C ) Sequence traces of these products showing an in-frame fusion between FGFR3 exon 18 and BAIAP2L1 exon 2. The junction is marked with an arrowhead. ( D ) RT112 cDNA made using a poly-dT primer with a unique 5′ sequence generated products following RT-PCR with forward primers from FGFR3 and a reverse primer from the unique sequence. Sequencing showed TACC3 involvement, and RT-PCR using a TACC3 reverse primer gave a unique band for RT112 and a series of bands for RT4. Arrows indicate FGFR3:TACC3 fusion transcripts that were confirmed by sequencing. RT− and RT+ are reverse transcriptase negative control and transcribed cDNA, respectively. ( E ) Sequence traces of FGFR3–TACC3 RT-PCR products showing the fusion gene junctions to be between FGFR3 exon 18 and TACC3 exon 11 in RT112 (arrowhead), and between FGFR3 within intron 18 and TACC3 within exon 4 in RT4 (bar).

Article Snippet: Sub-confluent TERT-NHUC were removed from flasks by treatment with accutase (Sigma) and allowed to rest in full medium for at least 20 min before staining with monoclonal anti-human FGFR3-phycoerythrin antibody (R&D Systems) according to the manufacturer's instructions.

Techniques: Clone Assay, Translocation Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Generated, Reverse Transcription, Negative Control

Journal: Human Molecular Genetics

Article Title: Oncogenic FGFR3 gene fusions in bladder cancer

doi: 10.1093/hmg/dds486

Figure Lengend Snippet: Schematic of FGFR3, TACC3 and BAIAP2L1 proteins showing known domains and positions of the breakpoints (black arrowheads) identified in bladder tumours (T1 and T2) and cell lines and those reported in glioblastoma (grey arrowheads) .

Article Snippet: Sub-confluent TERT-NHUC were removed from flasks by treatment with accutase (Sigma) and allowed to rest in full medium for at least 20 min before staining with monoclonal anti-human FGFR3-phycoerythrin antibody (R&D Systems) according to the manufacturer's instructions.

Techniques:

Journal: Human Molecular Genetics

Article Title: Oncogenic FGFR3 gene fusions in bladder cancer

doi: 10.1093/hmg/dds486

Figure Lengend Snippet: ( A ) Western blot shows the presence of a high-molecular weight form of FGFR3 (arrowheads) in the tumour from which LUCC2 was derived (T1), and that it is the same size as that seen in RT112 (arrowheads), with and without deglycosylation. ( B ) Immunoprecipitation of LUCC2 protein with antibodies detecting epitopes in the extracellular (E) or cytoplasmic (C) regions of FGFR3 and detection with FGFR3 antibody B9, which recognizes amino acids 25–124. A high-molecular weight form (arrowhead) is precipitated by E but not C antibody. Because of the lower FGFR3 expression, 20 times more total protein was used for the LUCC2 IP than for the cell lines shown in Fig. . ( C ) RT-PCR using primers from FGFR3 exon 18 and TACC3 exon 13 shows fusion transcripts in tumour samples T1 and T2 and cell lines RT4, RT112 and LUCC2. ( D ) Tumours T2, T10 and T11 retain high-molecular weight forms of FGFR3 after deglycosylation (arrowheads). Tumour T12 shows a high-molecular weight form of FGFR3 (arrowhead), but this is reduced to the normal molecular weight by deglycosylation. Cell line BFTC905 shows the expected result for untranslocated FGFR3.

Article Snippet: Sub-confluent TERT-NHUC were removed from flasks by treatment with accutase (Sigma) and allowed to rest in full medium for at least 20 min before staining with monoclonal anti-human FGFR3-phycoerythrin antibody (R&D Systems) according to the manufacturer's instructions.

Techniques: Western Blot, High Molecular Weight, Derivative Assay, Immunoprecipitation, Expressing, Reverse Transcription Polymerase Chain Reaction, Molecular Weight

Journal: Human Molecular Genetics

Article Title: Oncogenic FGFR3 gene fusions in bladder cancer

doi: 10.1093/hmg/dds486

Figure Lengend Snippet: Non-denaturing gel showing the presence of constitutively dimerized S249C mutant protein in 94-10 and 97-7 cells which contain point mutant (S249C) FGFR3 but not in NHUC, RT4 or SW780. A small amount of the dimer is present in RT112 (arrow).

Article Snippet: Sub-confluent TERT-NHUC were removed from flasks by treatment with accutase (Sigma) and allowed to rest in full medium for at least 20 min before staining with monoclonal anti-human FGFR3-phycoerythrin antibody (R&D Systems) according to the manufacturer's instructions.

Techniques: Mutagenesis

Journal: Human Molecular Genetics

Article Title: Oncogenic FGFR3 gene fusions in bladder cancer

doi: 10.1093/hmg/dds486

Figure Lengend Snippet: ( A ) Morphological changes in NIH-3T3 cells induced by ectopic expression of SW780FUS, RT112FUS and RT4 full-length (RT4FUS) fusion genes, but not with RT4-truncated form (RT4del), FGFR3IIIb, FGFR3 with exon 19 deleted (del19) or vector control. ( B ) Number of anchorage-independent colonies > 100 µm diameter in 15 × 1 mm 2 areas formed by NIH-3T3 cells expressing FGFR3 fusion proteins and controls. ( C ) NIH-3T3 cells expressing fusion genes and control constructs were serum starved for 1 h, immunoprecipitated with FGFR3 extracellular antibody E and detected using FGFR3 antibody, which recognizes amino acids 25–124. The presence of FGFR3 of the appropriate size is detected in each case; anti-phosphotyrosine antibody (middle panel) shows constitutive phosphorylation of RT112FUS, SW780FUS and RT4FUS fusion genes. Chart (lower panel) shows the relative intensities of signal from the two antibodies. ( D ) Fold increase in phospho-FGFR3 following stimulation with FGF1, presented as phospho-FGFR3:total FGFR3 following 10 min of FGF1 stimulation. Error bars are ± 1 SE.

Article Snippet: Sub-confluent TERT-NHUC were removed from flasks by treatment with accutase (Sigma) and allowed to rest in full medium for at least 20 min before staining with monoclonal anti-human FGFR3-phycoerythrin antibody (R&D Systems) according to the manufacturer's instructions.

Techniques: Expressing, Plasmid Preparation, Control, Construct, Immunoprecipitation, Phospho-proteomics

Journal: Human Molecular Genetics

Article Title: Oncogenic FGFR3 gene fusions in bladder cancer

doi: 10.1093/hmg/dds486

Figure Lengend Snippet: ( A ) Western blots showing levels of phospho-phospholipase C gamma 1 (PLCγ1) and phospho extracellular signal-regulated kinase (ERK) in TERT-NHUC expressing FGFR3 fusion proteins and controls. pFB, vector control; TERT-NHUC S249C and 97-7 cell line containing point-mutated FGFR3 (S249C) are positive controls. The PLCγ1 images are obtained from a single blot with some lanes removed. ( B ) Growth curves of TERT-NHUC expressing FGFR3 fusion proteins and controls. Cells expressing S249C-FGFR3 reach a higher confluent density than those expressing FGFR3–TACC3 fusion (RT112FUS) and controls. Error bars are ± 1 SE.

Article Snippet: Sub-confluent TERT-NHUC were removed from flasks by treatment with accutase (Sigma) and allowed to rest in full medium for at least 20 min before staining with monoclonal anti-human FGFR3-phycoerythrin antibody (R&D Systems) according to the manufacturer's instructions.

Techniques: Western Blot, Expressing, Plasmid Preparation, Control

Journal: International Journal of Molecular Sciences

Article Title: Profiling the Biological Characteristics and Transitions through Upper Tract Tumor Origin, Bladder Recurrence, and Muscle-Invasive Bladder Progression in Upper Tract Urothelial Carcinoma

doi: 10.3390/ijms23095154

Figure Lengend Snippet: ( a ) Representative immunohistochemistry (IHC) staining of FGFR3, p53, CK5/6, and CK20 in surgical specimens; pTa low-grade upper tract urothelial carcinoma (UTUC) patients (upper panel) and pT3 high-grade UTUC patients (lower panel). The field scale bar = 200 µm. ( b ) Heatmap describing the IHC score of FGFR3, p53, CK5/6, and CK20 in UTUC patients ( n = 214). Heatmap is divided by pT2 or low ( n = 116) and pT3 or high ( n = 98). The histoscore (H-score) was calculated by applying the following formula: mean percentage × intensity (range 0–300). ( c ) Table describing the rate of high expression group in each molecular biomarker. Expression rates were compared between patients who developed intravesical recurrence (IVR) and who did not develop IVR after radical nephroureterectomy (RNU). Chi-squared test was used to assess the significance of differences. ( d ) K–M curves comparing the IVR free survival rates in UTUC patients compared by low versus high FGFR3 expression, low versus high p53 expression, low versus high CK5/6 expression, and low versus high CK20 expression. Log-rank test was used to assess the significance of differences. ( e ) A schema describing the molecular characteristics of UTUC that are likely to develop IVR. As the multivariate analysis revealed, CK20 high and CK5/6 low expression tumor specimens were the independent risk factors for developing IVR after radical nephroureterectomy.

Article Snippet: The sections were then incubated at 4 °C overnight with anti-FGFR3 mouse monoclonal antibody (Ab; 1:150 dilution; Origene, Rockville, MD, USA), anti-p53 mouse monoclonal Ab (1:2000 dilution; Dako, Santa Clara, CA, USA), anti-CK5/6 mouse monoclonal Ab (1:100, Invitrogen, Waltham, MA, USA), and anti-CK20 mouse monoclonal Ab (1:100, Abcam, Cambridge, UK).

Techniques: Immunohistochemistry, Expressing, Biomarker Discovery

Journal: International Journal of Molecular Sciences

Article Title: Profiling the Biological Characteristics and Transitions through Upper Tract Tumor Origin, Bladder Recurrence, and Muscle-Invasive Bladder Progression in Upper Tract Urothelial Carcinoma

doi: 10.3390/ijms23095154

Figure Lengend Snippet: Uni- and multivariate Cox regression analyses for determining risk factors for IVR development in UTUC patients. Abbreviations: IVR; intravesical recurrence, UTUC; upper tract urothelial carcinoma, HR; hazard ratio, CI; confidence interval, UC; urothelial carcinoma, CIS; carcinoma in situ, LVI; lymphovascular invasion, FGFR; fibroblast growth factor receptor, CK; cytokeratin.

Article Snippet: The sections were then incubated at 4 °C overnight with anti-FGFR3 mouse monoclonal antibody (Ab; 1:150 dilution; Origene, Rockville, MD, USA), anti-p53 mouse monoclonal Ab (1:2000 dilution; Dako, Santa Clara, CA, USA), anti-CK5/6 mouse monoclonal Ab (1:100, Invitrogen, Waltham, MA, USA), and anti-CK20 mouse monoclonal Ab (1:100, Abcam, Cambridge, UK).

Techniques: In Situ, Adjuvant, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Profiling the Biological Characteristics and Transitions through Upper Tract Tumor Origin, Bladder Recurrence, and Muscle-Invasive Bladder Progression in Upper Tract Urothelial Carcinoma

doi: 10.3390/ijms23095154

Figure Lengend Snippet: ( a ) Representative IHC staining of FGFR3, p53, CK5/6, and CK20 in surgical specimens after transurethral resection of bladder tumor (TURBT); pTa low-grade IVR specimen, pT1 high-grade IVR specimen, and pT2 high-grade IVR specimen are shown, respectively. Low-power field scale bar = 500 µm, and high-power field scale bar = 100 µm. ( b ) Heatmap describing the IHC score of FGFR3, p53, CK5/6, and CK20 in transurethral resection (TUR) specimens after IVR ( n = 94). Heatmap is divided by pTa ( n = 35), pT1 ( n = 41), and pT2 or higher ( n = 18). The H-score was calculated by applying the following formula: mean percentage × intensity (range 0–270). ( c ) Graph showing the transition of four molecular markers’ expressions classified with pathological T stage. P53 and CK5/6 showed significantly higher expressions at muscle-invasive stage (pT2) than that of non-muscle-invasive stage (pTa and/or pT1; p = 0.001, p = 0.035), while CK20 expression decreased as the tumor stage progressed to MIBC ( p = 0.072). No significant differences were observed in FGFR3 expression along with tumor stage progression ( p = 0.822). ( d ) K–M curves comparing the bladder cancer progression free survival rates in UTUC patients who developed IVR compared by low versus high FGFR3 expression, low versus high p53 expression, low versus high CK5/6 expression, and low versus high CK20 expression. Log-rank test was used to assess the significance of differences. ( e ) A schema describing the transition of molecular characteristics of IVR specimen. At the time of bladder tumor recurrence, CK20 high and CK5/6 low expressions (luminal-like) occupy the majority of IVR specimen. In the process of MIBC progression, however, the high CK20 expression rate decreased as it progressed to muscle invasive, whereas the high CK5/6 expression rate increased (basal-like). Schema suggests the concept of “luminal–basal shift” with p53 addition from low stage bladder tumor recurrence to high stage bladder tumor progression.

Article Snippet: The sections were then incubated at 4 °C overnight with anti-FGFR3 mouse monoclonal antibody (Ab; 1:150 dilution; Origene, Rockville, MD, USA), anti-p53 mouse monoclonal Ab (1:2000 dilution; Dako, Santa Clara, CA, USA), anti-CK5/6 mouse monoclonal Ab (1:100, Invitrogen, Waltham, MA, USA), and anti-CK20 mouse monoclonal Ab (1:100, Abcam, Cambridge, UK).

Techniques: Immunohistochemistry, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Profiling the Biological Characteristics and Transitions through Upper Tract Tumor Origin, Bladder Recurrence, and Muscle-Invasive Bladder Progression in Upper Tract Urothelial Carcinoma

doi: 10.3390/ijms23095154

Figure Lengend Snippet: Uni- and multivariate Cox regression analyses for determining risk factors for MIBC progression after IVR development. Abbreviations: MIBC; muscle invasive bladder cancer, IVR; intravesical recurrence, HR; hazard ratio, CI; confidence interval, UC; urothelial carcinoma, CIS; carcinoma in situ, BT: bladder tumor, FGFR; fibroblast growth factor receptor, CK; cytokeratin.

Article Snippet: The sections were then incubated at 4 °C overnight with anti-FGFR3 mouse monoclonal antibody (Ab; 1:150 dilution; Origene, Rockville, MD, USA), anti-p53 mouse monoclonal Ab (1:2000 dilution; Dako, Santa Clara, CA, USA), anti-CK5/6 mouse monoclonal Ab (1:100, Invitrogen, Waltham, MA, USA), and anti-CK20 mouse monoclonal Ab (1:100, Abcam, Cambridge, UK).

Techniques: In Situ, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Profiling the Biological Characteristics and Transitions through Upper Tract Tumor Origin, Bladder Recurrence, and Muscle-Invasive Bladder Progression in Upper Tract Urothelial Carcinoma

doi: 10.3390/ijms23095154

Figure Lengend Snippet: ( a ) Representative IHC staining of FGFR3, p53, CK5/6, and CK20 in surgical specimens after radical cystectomy (RC). pT2 UTUC metachronous MIBC and pT2 bladder primary MIBC are shown. Power field scale bar = 200 µm. ( b ) Heatmap describing the IHC score of each molecular marker in MIBC patients who underwent RC ( n = 70). The heatmap of MIBC patients is classified by UTUC metachronous or bladder primary MIBC with information for age, sex, pT stage, grade, pN stage, histological type, and presence of LVI. ( c ) Chart describing the high/low expressions of all four molecular markers compared between UTUC metachronous and bladder primary MIBC. There were no significances in CK5/6, CK20, and p53 expressions between the two, while significantly higher FGFR3 expression was observed in UTUC metachronous MIBC compared to that of bladder primary MIBC. ( d ) K–M curves comparing the cancer-specific survival (CSS) rates in MIBC patients after RC compared by low versus high FGFR3 expression, low versus high p53 expression, low versus high CK5/6 expression, and low versus high CK20 expression. Log-rank test was used to assess the significance of differences. High p53 expression groups showed significantly lower CSS rates compared to the counterparts ( p = 0.037). In contrast, high FGFR3 expression group showed higher CSS rates compared to the counterparts.

Article Snippet: The sections were then incubated at 4 °C overnight with anti-FGFR3 mouse monoclonal antibody (Ab; 1:150 dilution; Origene, Rockville, MD, USA), anti-p53 mouse monoclonal Ab (1:2000 dilution; Dako, Santa Clara, CA, USA), anti-CK5/6 mouse monoclonal Ab (1:100, Invitrogen, Waltham, MA, USA), and anti-CK20 mouse monoclonal Ab (1:100, Abcam, Cambridge, UK).

Techniques: Immunohistochemistry, Marker, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Profiling the Biological Characteristics and Transitions through Upper Tract Tumor Origin, Bladder Recurrence, and Muscle-Invasive Bladder Progression in Upper Tract Urothelial Carcinoma

doi: 10.3390/ijms23095154

Figure Lengend Snippet: Uni- and multivariate analyses evaluating the prognostic factors associated with oncological outcomes in MIBC patients ( n = 70). Abbreviations: MIBC; muscle-invasive bladder cancer, HR; hazard ratio, CI; confidence interval, UTUC; upper tract urothelial carcinoma, LVI; lymphovascular invasion, FGFR; fibroblast growth factor receptor, CK; cytokeratin.

Article Snippet: The sections were then incubated at 4 °C overnight with anti-FGFR3 mouse monoclonal antibody (Ab; 1:150 dilution; Origene, Rockville, MD, USA), anti-p53 mouse monoclonal Ab (1:2000 dilution; Dako, Santa Clara, CA, USA), anti-CK5/6 mouse monoclonal Ab (1:100, Invitrogen, Waltham, MA, USA), and anti-CK20 mouse monoclonal Ab (1:100, Abcam, Cambridge, UK).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Profiling the Biological Characteristics and Transitions through Upper Tract Tumor Origin, Bladder Recurrence, and Muscle-Invasive Bladder Progression in Upper Tract Urothelial Carcinoma

doi: 10.3390/ijms23095154

Figure Lengend Snippet: ( a ) Heatmap describing the relationships of FGFR3 and p53 expressions from UTUC tumor origin to MIBC progression. Gray bar shows both FGFR3 and p53 low group. Light blue bar shows FGFR3 high but p53 low group. Yellow-green bar shows FGFR3 low but p53 high group. Purple bar shows FGFR3 high p53 high group. Table chart shows the distributions of FGFR3 and p53 expression rates among UTUC tumor origin ( n = 214), UTUC tumor origin limited to patients who developed IVR ( n = 94), IVR tumor specimen ( n = 94), and IVR tumor specimen that progressed to MIBC ( n = 18). ( b ) Heatmap describing the relationships of FGFR3 and p53 expression in UTUC metachronous MIBC ( n = 12) and bladder primary MIBC specimens ( n = 58). Table chart shows the distributions of FGFR3 and p53 expression rates of the two. ( c ) K–M curves showing the CSS rates in UTUC patients after RNU (on the left) and MIBC patients after RC (on the right), classified by four groups: FGFR3 low/p53 low (gray line), FGFR3 high/p53 low (blue line), FGFR3 low/p53 high (green line), and FGFR3 high/p53 high (purple line). ( d ) Schema showing the entire biological characteristics and transitions of four molecular markers in UTUC patients. UTUC patients with luminal-like dependent features are likely to develop IVR, and the molecular characteristics are mostly taken over to IVR specimen. After bladder tumor recurrence, the molecular characteristics shift from luminal-like to basal-like features as the tumor progresses to muscle-invasive status. Although p53 and CK5/6 show higher expression in the MIBC stage, UTUC metachronous MIBC maintains higher FGFR3 expression compared with bladder primary MIBC, which may comprise favorable biological features after RC.

Article Snippet: The sections were then incubated at 4 °C overnight with anti-FGFR3 mouse monoclonal antibody (Ab; 1:150 dilution; Origene, Rockville, MD, USA), anti-p53 mouse monoclonal Ab (1:2000 dilution; Dako, Santa Clara, CA, USA), anti-CK5/6 mouse monoclonal Ab (1:100, Invitrogen, Waltham, MA, USA), and anti-CK20 mouse monoclonal Ab (1:100, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Nature Communications

Article Title: Fibroblast growth factor 18 alleviates stress-induced pathological cardiac hypertrophy in male mice

doi: 10.1038/s41467-023-36895-1

Figure Lengend Snippet: a The corresponding Src family kinases correlated with the non-hypertrophic myocardium and hypertrophic myocardium were visualized by the heatmap in the normal and ISO treatment samples in GSE18801 dataset. The darker shade of red or blue represents the higher correlation level. b Circle plot depicting the important signal pathways associated with related genes. c NRCMs were treated with ISO (10 μM) in the presence or absence of FGF18 (50 ng/ml) for 48 h. Protein bands at ~60 kDa were excised from the SDS-PAGE gels for LC-MS/MS analysis. d NRCMs were treated with FGF18 (50 ng/ml) for 10 min. The cell lysates were subjected to immunoprecipitation with FGFR3 antibody, followed by immunoblotting with the FYN antibody. Cell lysates were also subjected to immunoprecipitation with IgG as negative control. n = 4. e NRCMs were treatment with FGF18 (50 ng/ml) for 10 min. The cell lysates were subjected to immunoprecipitation with FYN antibody, followed by immunoblotting with the p-SrcY416 and FGFR3 antibodies. Cell lysates were also subjected to immunoprecipitation with IgG as a negative control. n = 4. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source data file.

Article Snippet: For RNA interference, cells were transfected with FGFR3 siRNA (Origene, SR513507) or control scrambled siRNA (Origene, SR30004) by Lipofectamine 3000 for 12 h in Opti-MEM.

Techniques: SDS Page, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Western Blot, Negative Control

Journal: Nature Communications

Article Title: Fibroblast growth factor 18 alleviates stress-induced pathological cardiac hypertrophy in male mice

doi: 10.1038/s41467-023-36895-1

Figure Lengend Snippet: FGF18/FGFR3 promoted the activity and expression of FYN and negatively regulated NOX4 activity, thereby inhibiting ROS generation and protecting heart function by inhibiting cardiac fibrosis and hypertrophy. In conclusion, the present study identifies a previously unknown biological function of FGF18 in pathological cardiac hypertrophy. Part of the figure is modified from Servier Medical Art ( http://smart.servier.com/ ), licensed under a Creative Common Attribution 3.0 unported License.

Article Snippet: For RNA interference, cells were transfected with FGFR3 siRNA (Origene, SR513507) or control scrambled siRNA (Origene, SR30004) by Lipofectamine 3000 for 12 h in Opti-MEM.

Techniques: Activity Assay, Expressing, Modification