Journal: The Journal of Clinical Investigation

Article Title: Mutated FGFR1 is an oncogenic driver and therapeutic target in high-risk neuroblastoma

doi: 10.1172/JCI189152

Figure Lengend Snippet: ( A ) Relative cell counts of viable Ba/F3 cells transduced with different vectors (empty vector, FGFR1 WT, FGFR1 D623A , FGFR1 D623A;N546K , FGFR1 N546K ) normalized to parental Ba/F3 cells. Analyses were performed with 10 ng/mL IL-3 in culture medium and after withdrawal of IL-3 after 144 hours. Average numbers (±SD) of 6 independent experiments are shown. P values were calculated in pair-wise comparisons to Ba/F3 parental cells using a 1-sided Wilcoxon rank-sum test and adjusted for multiple testing using the Benjamini–Hochberg method. ( B ) Levels of total and phosphorylated proteins of the FGFR pathway in IL-3–dependent Ba/F3 cells (parental, empty vector, FGFR1 WT, FGFR1 D623A , FGFR1 D623A;N546K , FGFR1 N546K ) and 3 IL-3–independent FGFR1 N546K - transduced Ba/F3 cell clones (blue). IL-3–independent FGFR1 N546K - transduced BA/F3 cells shown in different blue tones correspond to cells from 3 independent experiments labelled in light blue in ( A ). Antibodies against total and phosphorylated FGFR1, the adaptor protein FRS2, and the downstream targets AKT, ERK, and STAT3 were used. β-actin served as loading control.

Article Snippet: The R26-LSL-FGFR1 N546K genetically engineered mouse line was generated by Taconic Biosciences and crossbred with the established Th-MYCN and Th-IRES-Cre mouse lines ( , ).

Techniques: Transduction, Plasmid Preparation, Clone Assay, Control

Journal: The Journal of Clinical Investigation

Article Title: Mutated FGFR1 is an oncogenic driver and therapeutic target in high-risk neuroblastoma

doi: 10.1172/JCI189152

Figure Lengend Snippet: ( A ) Relative cell viability of IL-3–dependent Ba/F3 cells (parental, empty vector, FGFR1 WT, FGFR1 D623A , FGFR1 D623A;N546K , FGFR1 N546K ) and 3 IL-3–independent FGFR1 N546K - transduced Ba/F3 cell clones (blue) after treatment with various concentrations (conc.) of futibatinib (in μM: 0.0001, 0.001, 0.01, 0.0398, 0.0631, 0.1, 1, 10, and 100) and DMSO as control for 72 hours. Mean cell viabilities ± SD of 3 ( FGFR1 WT, FGFR1 D623A , FGFR1 D623A;N546K , FGFR1 N546K ) or 4 (parental, empty vector, FGFR1 N546K IL-3–independent cells) independent experiments conducted in triplicate each are plotted. ( B ) IC 50 of futibatinib in IL-3–dependent Ba/F3 cells and 3 IL-3–independent FGFR1 N546K -transduced Ba/F3 cell clones, derived from 3 ( FGFR1 WT, FGFR1 D623A , FGFR1 D623A;N546K , FGFR1 N546K ) or 4 (parental, empty vector, FGFR1 N546K IL-3–independent cells) independent experiments consisting of triplicates each. P values were calculated in pair-wise comparisons to Ba/F3 parental cells using a 1-sided Wilcoxon rank-sum test and adjusted for multiple testing using the Benjamini–Hochberg method. ( C ) Levels of total and phosphorylated proteins of the FGFR pathway in IL-3–dependent Ba/F3 cells (parental, empty vector, FGFR1 WT, FGFR1 D623A , FGFR1 D623A;N546K , FGFR1 N546K ) and 3 IL-3–independent FGFR1 N546K transduced Ba/F3 cell clones (blue) after treatment with DMSO or 10, 50, or 100 nM futibatinib (experiment was conducted 3 times).

Article Snippet: The R26-LSL-FGFR1 N546K genetically engineered mouse line was generated by Taconic Biosciences and crossbred with the established Th-MYCN and Th-IRES-Cre mouse lines ( , ).

Techniques: Plasmid Preparation, Clone Assay, Control, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Mutated FGFR1 is an oncogenic driver and therapeutic target in high-risk neuroblastoma

doi: 10.1172/JCI189152

Figure Lengend Snippet: ( A ) Images of abdominal cavities of R26-LSL-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt mice sacrificed at the ages of 7 and 21 days, showing tumors between the kidneys (top). Arrows point to the tumor boundaries. Kidneys (K) and tumors (indicated by asterisks) were prepared for better visualization of the tumors (bottom). ( B ) H&E and PHOX2B and Ki67 immunohistochemical staining of tumor sections obtained from R26-LSL-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt mice at the ages of 7 (left) and 14 (right) days; scale bar: 50 μm. ( C and D ) OS ( C ) and neuroblastoma-specific (NB-specific) survival ( D ) of R26-LSL-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt (blue), Th-MYCN tg/wt (red), and Th-ALK F1174L/wt (yellow) mice. Although OS was reduced in FGFR1 N546K transgenic mice in comparison with the other groups, due to development of papillomas and sarcomas ( FGFR1 N546K vs. MYCN tg , P < 0.001; FGFR1 N546K vs. ALK F1174L , P < 0.001; MYCN tg vs. ALK F1174L , P = 0.106), neuroblastoma-specific survival of FGFR1 N546K transgenic mice was worse only in comparison with ALK F1174L mice ( P = 0.049), but did not differ from that of MYCN tg mice ( P = 0.226). Survival of ALK F1174L compared with MYCN tg mice was not significant ( P = 0.394). Survival curves were estimated according to Kaplan-Meier test and compared with a log-rank test.

Article Snippet: The R26-LSL-FGFR1 N546K genetically engineered mouse line was generated by Taconic Biosciences and crossbred with the established Th-MYCN and Th-IRES-Cre mouse lines ( , ).

Techniques: Immunohistochemical staining, Staining, Transgenic Assay, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Mutated FGFR1 is an oncogenic driver and therapeutic target in high-risk neuroblastoma

doi: 10.1172/JCI189152

Figure Lengend Snippet: ( A ) Axial T2-weighted MRI scans of an R26-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt ;Th-MYCN tg/wt mouse that developed neuroblastomas at the adrenal glands (left) and the pelvic sympathetic trunk (right). ( B ) OS of R26-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt ;Th-MYCN tg/wt , Th- ALK F1174L/wt ; Th-MYCN tg/wt , and R26-LSL-FGFR1 N546K/wt ;Th-MYCN tg/wt mice. Survival of all groups differed significantly from each other in pairwise comparisons ( P < 0.001 each). Survival curves were estimated according to Kaplan-Meier tests and compared with log-rank tests. ( C ) H&E (top) and PHOX2B immunohistochemical (bottom) staining of an adrenal tumor obtained from an R26-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt ;Th-MYCN tg/wt mouse at the age of 21 days, and of an adrenal tumor obtained from a Th-ALK F1174L/wt ;Th-MYCN tg/wt mouse at the age of 49 days; scale bar: 50 μm. ( D ) FGFR1 transcript levels in tumors obtained from R26-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt ;Th-MYCN tg/wt mice and from Th-ALK F1174L/wt ;Th-MYCN tg/wt mice ( n = 11 each), as determined by RNA-Seq. Box plots show the median, first, and third quartiles; the whiskers represent the minimum and maximum values within ±1.5 times the interquartile range. Comparisons between groups were performed using a 2-tailed Wilcoxon rank-sum test.

Article Snippet: The R26-LSL-FGFR1 N546K genetically engineered mouse line was generated by Taconic Biosciences and crossbred with the established Th-MYCN and Th-IRES-Cre mouse lines ( , ).

Techniques: Immunohistochemical staining, Staining, RNA Sequencing

Journal: The Journal of Clinical Investigation

Article Title: Mutated FGFR1 is an oncogenic driver and therapeutic target in high-risk neuroblastoma

doi: 10.1172/JCI189152

Figure Lengend Snippet: ( A – C ) Expression levels of genes associated with proliferation ( Mki67 , Ccnb1 , Ccnd1 , Top2a , and Pcna ( A ); anti-apoptotic genes ( Bcl2 and Bcl2l1 ) ( B ); and pro-apoptotic genes ( Casp3, Bax , and Bid ) ( C ) in tumors of R26-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt ;Th-MYCN tg/wt and Th-ALK F1174L/wt ;Th-MYCN tg/wt mice, as determined by RNA-Seq. Box plots show the median, first, and third quartiles; the whiskers represent the minimum and maximum values within ±1.5 times the interquartile range. Comparisons between groups were performed using a 2-tailed Wilcoxon rank-sum test. ( D ) H&E and PHOX2B, Ki67, and cleaved caspase 3 (CC3) immunohistochemical staining of tumor sections obtained from a Th-ALK F1174L/wt ;Th-MYCN tg/wt mouse (56 days old; top) and from an R26-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt ;Th-MYCN tg/wt mouse (20 days old; bottom); scale bar, 50 μm. ( E ) BCL2 immunohistochemical staining of tumor sections obtained from of Th-ALK F1174L/wt ;Th-MYCN tg/wt (top) and R26-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt ;Th-MYCN tg/wt (bottom) mice; scale bar: 100 μm.

Article Snippet: The R26-LSL-FGFR1 N546K genetically engineered mouse line was generated by Taconic Biosciences and crossbred with the established Th-MYCN and Th-IRES-Cre mouse lines ( , ).

Techniques: Expressing, RNA Sequencing, Immunohistochemical staining, Staining

Journal: The Journal of Clinical Investigation

Article Title: Mutated FGFR1 is an oncogenic driver and therapeutic target in high-risk neuroblastoma

doi: 10.1172/JCI189152

Figure Lengend Snippet: ( A ) Temporal changes of relative tumor diameters in R26-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt ;Th-MYCN tg/wt mice treated with 5 mg/kg futibatinib (light green), 30 mg/kg futibatinib (dark green), or control substance (0.5% CMC-Na; blue). Growth curves of individual tumors are shown on the left; mean and range of the relative tumor diameter changes are shown on the right. ( B ) OS of R26-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt ;Th-MYCN tg/wt mice treated with 5 mg/kg, or 30 mg/kg futibatinib, or control. Survival of mice treated with 5 mg/kg futibatinib was significantly longer than that of control mice ( P < 0.001), whereas survival of mice treated with 30 mg/kg futibatinib was not significantly prolonged, due to toxicity ( P = 0.47). ( C ) Axial T2-weighted MRI scans of an R26-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt ;Th-MYCN tg/wt mouse treated with 5 mg/kg futibatinib at the indicated ages. ( D ) H&E and PHOX2B, Ki67, and CC3 immunohistochemical staining of tumor sections obtained from R26-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt ;Th-MYCN tg/wt mice treated with control (top) or 5 mg/kg futibatinib (bottom); scale bar, 50 μm. ( E ) Absolute volumes of tumors obtained from R26-FGFR1 N546K/wt ;Th-IRES-Cre tg/wt ;Th-MYCN tg/wt mice, subcutaneously reimplanted into NSG mice and treated with 10 mg/kg futibatinib ( n = 11) or control ( n = 9) after reaching a volume of 0.08–0.2 cm 3 . Growth curves of individual tumors are shown on the left; mean and range of tumor volumes are shown on the right.

Article Snippet: The R26-LSL-FGFR1 N546K genetically engineered mouse line was generated by Taconic Biosciences and crossbred with the established Th-MYCN and Th-IRES-Cre mouse lines ( , ).

Techniques: Control, Immunohistochemical staining, Staining

Journal: The Journal of Clinical Investigation

Article Title: Mutated FGFR1 is an oncogenic driver and therapeutic target in high-risk neuroblastoma

doi: 10.1172/JCI189152

Figure Lengend Snippet: ( A ) Absolute volumes of individual tumors (left) and mean and range of tumor volumes (right) of an FGFR N546K -mutant, patient-derived xenograft mouse model, treated with control (10% DMSO + 90% [20%] Captisol in 0.9% NaCl); n = 13) or 20 mg/kg futibatinib (futibatinib diluted in 10% DMSO + 90% [20%] Captisol in 0.9% NaCl); n = 13). ( B ) OS of mice bearing an FGFR N546K -mutant, patient-derived xenograft, treated with control or 20 mg/kg futibatinib. Survival curves were estimated according to Kaplan-Meier test and compared with a log-rank test. ( C ) H&E and PHOX2B and Ki67 immunohistochemical staining of tumor sections obtained from patient-derived xenograft tumors treated with a control substance (top) of 20 mg/kg futibatinib (bottom); scale bar, 100 μm.

Article Snippet: The R26-LSL-FGFR1 N546K genetically engineered mouse line was generated by Taconic Biosciences and crossbred with the established Th-MYCN and Th-IRES-Cre mouse lines ( , ).

Techniques: Mutagenesis, Derivative Assay, Control, Immunohistochemical staining, Staining

Journal: The Journal of Clinical Investigation

Article Title: Mutated FGFR1 is an oncogenic driver and therapeutic target in high-risk neuroblastoma

doi: 10.1172/JCI189152

Figure Lengend Snippet: ( A ) Schematic timeline of the clinical course of a patient with FGFR1 N546K -mutated neuroblastoma that progressed under multiple lines of treatment and therefore was treated with a combination of futibatinib, cyclophosphamide, and topotecan. ( B ) Computed tomography (CT) scans of the tumor region in patient shown in ( D ) on day 15 after start of futibatinib/cyclophosphamide/topotecan (day 380 after diagnosis) and on day 87 (day 442 after diagnosis), demonstrating a partial regression of the tumor. ( C ) Absolute volumes of individual tumors (left) and mean and range of tumor volumes (right) of the patient-derived xenograft mouse model, treated with cyclophosphamide/topotecan alone (blue) or in combination with 20 mg/kg futibatinib (green). ( D ) OS of mice bearing the patient-derived xenograft, treated with cyclophosphamide/topotecan alone (blue) or in combination with 20 mg/kg futibatinib (green). Survival curves were estimated according to Kaplan-Meier test and compared with a log-rank test. MIBG, metaiodobenzylguanidine.

Article Snippet: The R26-LSL-FGFR1 N546K genetically engineered mouse line was generated by Taconic Biosciences and crossbred with the established Th-MYCN and Th-IRES-Cre mouse lines ( , ).

Techniques: Computed Tomography, Biomarker Discovery, Derivative Assay

Journal: Cells

Article Title: GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells toward Breast Tumor Progression

doi: 10.3390/cells8030223

Figure Lengend Snippet: Analysis of METABRIC and TCGA datasets and CRISPR/Cas9-mediated FGFR1 knockout (KO) in MDA-MB-231 cells. ( a , b ) The OncoPrint of genomic alteration of FGFRs members showed that FGFR1 is the most amplified receptor of the family in human breast cancer patients. Each row represents a gene and each column represents a tumor sample. Red bars indicate gene amplifications, blue bars deep deletions and grey bars no alterations. ( c , d ) Kaplan-Meir plots show the overall survival (OS) from METABRIC dataset between patients with normal or high FGFR1 mRNA expression or between patients with copy number (CN) gains or without (neutral). Statistical analysis was performed using the long-rank test. ( e ) Schematic representation of the pX459 plasmid and the sgRNA sequence used to generate FGFR1 (KO) MDA-MB-231 cells. ( f ) Immunoblots of lysates generated from FGFR1 (WT) and FGFR1 (KO) MDA-MB-231 cells. β-actin served as loading control. Immunoblots shown are representative of three independent experiments.

Article Snippet: Primary antibodies were as follows: GPER (AB137479) (Abcam, Euroclone Milan, Italy); CTGF (TA806803) (OriGene Technologies, DBA, Milan, Italy); FGFR1 (#9740) and p-FGFR1 (#3476) (CST, Euroclone Milan, Italy); c-fos (E8), phosphorylated extracellular signal-regulated kinase (ERK) (E-4), ERK2 (C-14), p-AKT1/2/3 (Ser 473)-R, AKT/1/2/3 (H-136) and β-actin (AC-15) (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: CRISPR, Knock-Out, Amplification, Expressing, Plasmid Preparation, Sequencing, Western Blot, Generated

Journal: Cells

Article Title: GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells toward Breast Tumor Progression

doi: 10.3390/cells8030223

Figure Lengend Snippet: Conditioned medium (CM) from estrogen-stimulated CAFs induces the activation of FGFR1- signaling pathway in MDA-MB-231 cells. ( a – c ) Phosphorylation of FGFR1, ERK1/2, AKT in MDA-MB-231 cells exposed for 1 h to CM from CAFs treated for 18 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)], alone and in the presence of 1 μM FGFR1 inhibitor PD173074. ( d , e ) Activation of ERK1/2 and AKT in FGFR1 (WT) MDA-MB-231 cells upon exposure for 1 h to CM from CAFs treated for 18 h with 10 nM E2 [CM/CAFs (+E2)], 100 nM G-1 [CM/CAFs (+G-1)]; ( f , g ) In FGFR1 (KO) MDA-MB-231 cells cultured in the same conditions as described above, the activation of ERK1/2 and AKT was no longer observed. FGF2 at 25 nM was used as positive control. FGFR1, ERK2, AKT and β-actin served as loading control, as indicated. Side panels show densitometric analysis of the blots normalized to the loading controls. Immunoblots shown are representative of three independent experiments. (*) indicates p < 0.05.

Article Snippet: Primary antibodies were as follows: GPER (AB137479) (Abcam, Euroclone Milan, Italy); CTGF (TA806803) (OriGene Technologies, DBA, Milan, Italy); FGFR1 (#9740) and p-FGFR1 (#3476) (CST, Euroclone Milan, Italy); c-fos (E8), phosphorylated extracellular signal-regulated kinase (ERK) (E-4), ERK2 (C-14), p-AKT1/2/3 (Ser 473)-R, AKT/1/2/3 (H-136) and β-actin (AC-15) (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Activation Assay, Cell Culture, Positive Control, Western Blot

Journal: Cells

Article Title: GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells toward Breast Tumor Progression

doi: 10.3390/cells8030223

Figure Lengend Snippet: Conditioned medium (CM) from estrogen-stimulated CAFs up-regulates CTGF levels through FGFR1 signaling pathway in MDA-MB-231 cells. ( a ) Pairwise linear regressions of FGFR1 versus CTGF mRNA levels were performed on METABRIC dataset of 1904 breast tumor samples. Scatter plot shows positive correlation between FGFR1 and CTGF expression. ( b – d ) CTGF mRNA and protein levels in FGFR1 (WT) and FGFR1 (KO) MDA-MB-231 cells exposed for 3 h to CM from CAFs treated for 18 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)], or exposed to 25 nM FGF2, as positive control, evaluated by qPCR and western blot. In RNA experiments, values were normalized to the expression of 18S and shown as fold changes of CTGF mRNA expression upon CM from CAFs treated with E2 and G-1 respect to cells exposed to CM from CAFs treated with vehicle. Each column represents the mean ±SD of three independent experiments performed in triplicate. ( e – g ) Up-regulation of CTGF protein expression in MDA-MB-231 cells exposed for 3 h to CM from CAFs treated for 18 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)], or 100 nM G-1 [CM/CAFs (+G-1)] was no longer observed in the presence of 1 μM FGFR1 inhibitor PD173074, 10 μM MEK inhibitor PD98059 or 100 nM PI3K inhibitor Wortmannin (WM). β-actin served as loading control. Side panels show densitometric analysis of the blots normalized to the loading controls. Immunoblots shown are representative of three independent experiments. (**) indicates p < 0.01 and (*) indicates p < 0.05.

Article Snippet: Primary antibodies were as follows: GPER (AB137479) (Abcam, Euroclone Milan, Italy); CTGF (TA806803) (OriGene Technologies, DBA, Milan, Italy); FGFR1 (#9740) and p-FGFR1 (#3476) (CST, Euroclone Milan, Italy); c-fos (E8), phosphorylated extracellular signal-regulated kinase (ERK) (E-4), ERK2 (C-14), p-AKT1/2/3 (Ser 473)-R, AKT/1/2/3 (H-136) and β-actin (AC-15) (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Expressing, Positive Control, Western Blot

Journal: Cells

Article Title: GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells toward Breast Tumor Progression

doi: 10.3390/cells8030223

Figure Lengend Snippet: FGFR1 paracrine activation promotes migration and invasion in MDA-MB-231 cells. ( a ) FGFR1 (WT) and (b ) FGFR1 (KO) MDA-MB-231 cells were cultured for 8 h in CM from CAFs treated for 18 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)], or exposed to 25 nM FGF2, as positive control. Lines traced on cells were used to calculate the Polarity Index (PI). White lines define the migratory axis and black lines the transversal axis. PI = 1.0 indicates a polygonal shape, whereas a value > 1.0 defines ranges of migratory shapes. Scale bar = 30 μm. Images shown are representative of 30 random fields acquired in three independent experiments. Transwell assays were used to assess cell migration ( c ) and invasion ( d ) in FGFR1 (WT) and FGFR1 (KO) MDA-MB-231 cells cultured for 8 h in CM from CAFs treated for 18 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)], or exposed to 25 nM FGF2, as positive control. Cells were counted in at least 10 random fields at 10× magnification, in three independent experiments performed in triplicate. Scale bar = 200 μm, (**) indicates p < 0.01.

Article Snippet: Primary antibodies were as follows: GPER (AB137479) (Abcam, Euroclone Milan, Italy); CTGF (TA806803) (OriGene Technologies, DBA, Milan, Italy); FGFR1 (#9740) and p-FGFR1 (#3476) (CST, Euroclone Milan, Italy); c-fos (E8), phosphorylated extracellular signal-regulated kinase (ERK) (E-4), ERK2 (C-14), p-AKT1/2/3 (Ser 473)-R, AKT/1/2/3 (H-136) and β-actin (AC-15) (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Activation Assay, Migration, Cell Culture, Positive Control

Journal: Cells

Article Title: GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells toward Breast Tumor Progression

doi: 10.3390/cells8030223

Figure Lengend Snippet: CTGF is required for migration and invasion induced by FGFR1 paracrine activation in MDA-MB-231 cells. Transwell assays were used to assess cell migration ( a ) and invasion ( b ) in MDA-MB-231 cells transfected for 24 h with control shRNA or shCTGF and then cultured for 8 h in CM from CAFs treated for 18 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)]. Cells were counted in at least 10 random fields at 10× magnification, in three independent experiments performed in triplicate. Scale bar = 200 μm, (**) indicates p < 0.01.

Article Snippet: Primary antibodies were as follows: GPER (AB137479) (Abcam, Euroclone Milan, Italy); CTGF (TA806803) (OriGene Technologies, DBA, Milan, Italy); FGFR1 (#9740) and p-FGFR1 (#3476) (CST, Euroclone Milan, Italy); c-fos (E8), phosphorylated extracellular signal-regulated kinase (ERK) (E-4), ERK2 (C-14), p-AKT1/2/3 (Ser 473)-R, AKT/1/2/3 (H-136) and β-actin (AC-15) (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Migration, Activation Assay, Transfection, shRNA, Cell Culture

Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a Schematic diagram of IGF1R and FGFR1 activation pathways. b , c Co-IP ( b ) and immunofluorescence ( c ) to detect the interaction between ARMH4 and IGF1R/FGFR1 in HEK293 cells co-transfected with pcDNA3.1-h Armh4 -3FLAG and pcDNA3.1-h Igf1r -HA, as well as pcDNA3.1-h Armh4 -3FLAG and pcDNA3.1-h Fgfr1 -HA, Scale bar, 10 μm. All experiments were performed twice independently with comparable results, and representative data from one experiment are shown. d , e Validation of ARMH4-IGF1R ( d ) and ARMH4-FGFR1 ( e ) interaction using endogenous IP in heart tissues. All experiments were performed twice independently with comparable results, and representative data from one experiment are shown. f AlphaFold 3 simulation of the effect of ARMH4 on ligand-receptor binding for IGF1/IGF1R and FGF2/FGFR1. g AlphaFold3 simulation of the specific binding region of ARMH4 within the IGF1/IGF1R ligand-receptor complex. h Schematic diagram of the designed plasmid construct targeting the predicted binding sequence between ARMH4 and IGF1R. i Co-IP analysis of the interaction between IGF1R and ARMH4, including its isoforms, in HEK293 cells. All experiments were performed twice independently with comparable results, and representative data from one experiment are shown. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Igf1r -HA and pcDNA3.1-h Fgfr1 -HA recombinant plasmids were constructed by cloning the coding region of h Igf1r and h Fgfr1 into the NheI and XbaI (NEB, #R0145) sites of the pcDNA3.1-HA plasmid. pLV3-CMV- Myc (mouse)-3FLAG-Fluc-Puro was purchased from MIAOLING BIOLOGY (#P58001).

Techniques: Activation Assay, Co-Immunoprecipitation Assay, Immunofluorescence, Transfection, Biomarker Discovery, Binding Assay, Plasmid Preparation, Construct, Sequencing

Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a , b Adenoviral overexpression of Armh4 with PPP ( a ) and FA ( b ) treatment in NRVMs followed by Western blot analysis of phosphorylated mTOR, p70S6K, Akt and ERK. Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. c , d Adenoviral overexpression of Armh4 with PPP ( c ) and FA ( d ) treatment in NRVMs followed by western blot to detect the autophagy-related markers. Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. e , f Adenoviral overexpression of Armh4 with siRNA-mediated knockdown of Igf1r ( e ) and Fgfr1 ( f ) in NRVMs followed by western blot analysis of phosphorylated p70S6K, Akt, ERK and LC3. Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. g , h Adenoviral overexpression of Armh4 with IGF1 ( g ) and FGF2 ( h ) treatment in NRVMs followed by western blot analysis of phosphorylated p70S6K, Akt, ERK and LC3. n = 3. Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by two-way ANOVA, and the exact P values are reported directly on the figures. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Igf1r -HA and pcDNA3.1-h Fgfr1 -HA recombinant plasmids were constructed by cloning the coding region of h Igf1r and h Fgfr1 into the NheI and XbaI (NEB, #R0145) sites of the pcDNA3.1-HA plasmid. pLV3-CMV- Myc (mouse)-3FLAG-Fluc-Puro was purchased from MIAOLING BIOLOGY (#P58001).

Techniques: Over Expression, Western Blot, Knockdown

Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a Western blot analysis of FGFR1, IGFR1 protein expression in the hearts of young and aged WT and KO mice. Representative blot of n = 3 independent mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 independent mice. Statistical significance was determined by two-way ANOVA. b Western blot analysis of FGFR1, IGFR1 protein expression in the livers of young and aged WT and KO mice. Representative blot of n = 3 independent mice. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 independent mice. Statistical significance was determined by two-way ANOVA. c Western blot analysis of FGFR1, IGFR1 protein expression in Armh4 -deficient MEFs. Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. d, e Western blot analysis of FGFR1 and IGFR1 protein expression in NRVMs with/without siRNA-mediated knockdown of Armh4 ( d ) and adenoviral overexpression of Armh4 ( e ). Representative blot of n = 3 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. f Western blot analysis of FGFR1 and IGFR1 protein expression in NRVMs treated with recombinant ARMH4. n = 6. Representative blot of n = 6 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 6 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. The exact P values are reported directly on the figures. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Igf1r -HA and pcDNA3.1-h Fgfr1 -HA recombinant plasmids were constructed by cloning the coding region of h Igf1r and h Fgfr1 into the NheI and XbaI (NEB, #R0145) sites of the pcDNA3.1-HA plasmid. pLV3-CMV- Myc (mouse)-3FLAG-Fluc-Puro was purchased from MIAOLING BIOLOGY (#P58001).

Techniques: Western Blot, Expressing, Two Tailed Test, Knockdown, Over Expression, Recombinant

Journal: Nature Communications

Article Title: ARMH4 accelerates aging by maintaining a positive-feedback growth signaling circuit

doi: 10.1038/s41467-025-67505-x

Figure Lengend Snippet: a TF-Target Finder predicted the transcription factors that co-regulated by IGF1R and FGFR1 screening diagram. b Western blot analysis of c-Myc protein expression in NRVMs upon Armh4 knockdown and overexpression. Representative blot of n = 6 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 6 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. c, d qRT-PCR of Myc expression in NRVMs upon Armh4 knockdown ( c ) and overexpression ( d ). Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. e Schematic diagram of the dual-luciferase reporter constructs. f Dual-luciferase reporter assay in HEK293 cells. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. g Screening the c-Myc binding motifs by JASPAR. h Computational prediction of c-Myc binding motifs in IGF1R/FGFR1 regulatory regions using JASPAR. i Western blot analysis of c-Myc, IGF1R, FGFR1 expression in NRVMs upon Myc knockdown by siRNA. Representative blot of n = 6 biological replicates. Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 6 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. j–m Western blot analysis of IGF1R and FGFR1 expression ( j ), phosphorylated p70S6K, Akt, ERK ( k ), puromycin incorporation ( l ), the autophagy-related marker ( m ) in NRVMs with Myc knockdown followed by Armh4 overexpression. Representative blot of n = 4 biological replicates ( j , k , m ) and n = 3 biological replicates ( l ). Data are presented as mean values +/- SEM. Black circles represent individual data points for n = 4 biological replicates ( j , k , m ) and n = 3 biological replicates ( l ). Statistical significance was determined by two-way ANOVA. The exact P values are reported directly on the figures. Source data are provided as a Source Data file.

Article Snippet: The pcDNA3.1-h Igf1r -HA and pcDNA3.1-h Fgfr1 -HA recombinant plasmids were constructed by cloning the coding region of h Igf1r and h Fgfr1 into the NheI and XbaI (NEB, #R0145) sites of the pcDNA3.1-HA plasmid. pLV3-CMV- Myc (mouse)-3FLAG-Fluc-Puro was purchased from MIAOLING BIOLOGY (#P58001).

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Two Tailed Test, Quantitative RT-PCR, Luciferase, Construct, Reporter Assay, Binding Assay, Marker

Journal: eLife

Article Title: Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation

doi: 10.7554/eLife.52027

Figure Lengend Snippet:

Article Snippet: After blocking with 5% non-fat dried milk in Tris-buffered saline Tween-20 (TBST; 28 mM Tris, 137 mM NaCl, 0.05% Tween-20, pH 7.4) for 45 min at room temperature, membranes were probed for 1 hr at room temperature or overnight at 4°C with mouse anti-phosphotyrosine (1:1000, Cell Signaling Technology 9411S), rabbit anti-Nlgn1 (1:1000, Synaptic systems 129013), rabbit anti-FGFR1 (1:1000, Cell Signaling Technology D8E4), or rat anti-HA (1:1000, Roche 3F10).

Techniques: Transfection, Construct, Cloning, Plasmid Preparation, Electroporation, shRNA, Recombinant, Sequencing, Immunoprecipitation, Software

Journal: Vascular Pharmacology

Article Title: Soluble N-cadherin: A novel inhibitor of VSMC proliferation and intimal thickening

doi: 10.1016/j.vph.2015.11.040

Figure Lengend Snippet: QPCR primers.

Article Snippet: Antibodies were used at the following concentrations: N-cadherin (BD Biosciences: 610920, 1:2500), GAPDH (Chemicon: MAB374, 1:5000), and FGFR1 (R&D Systems: MAB658, 1:50).

Techniques: Sequencing

Journal: Vascular Pharmacology

Article Title: Soluble N-cadherin: A novel inhibitor of VSMC proliferation and intimal thickening

doi: 10.1016/j.vph.2015.11.040

Figure Lengend Snippet: FGF-R was essential for the anti-proliferative effect of soluble N-cadherin. a: Proliferation (percentage of BrdU positive human VSMCs) following treatment with SU5402 (an inhibitor of FGFR1) and 100 pM Fc or SNC-Fc, in the presence of bFGF and PDGF for 24 h. * indicates a significant difference from the Fc control, n = 3. b: Proliferation (percentage of BrdU positive human VSMCs) following siRNA for FGFR1 and treatment with 100 pM Fc or SNC-Fc in the presence of bFGF and PDGF for 24 h. * indicates a significant difference from the relevant Fc control, n = 3. c: Proliferation (percentage of BrdU positive human VSMCs) following 24 h incubation with bFGF and PDGF and 100 pM Fc, SNC-Fc or SNC-Fc with either the N-cadherin binding site mutation (SNC-Fc N-cadmut) or FGF-R binding site mutated (SNC-Fc FGFRmut). * indicates a significant difference from the Fc control, n = 3. d: Western blot for Fc-tag in VSMCs overexpressing FGFR1GFP and incubated with Fc/SNC-Fc/SNC-Fc for 1 h, before immunoprecipitation for GFP tagged FGFR and Western blotting (i). Western blot for Fc-tag in VSMCs overexpressing full length N-cadherin and incubated with Fc/SNC-Fc/SNC-Fc FGFR for 1 h, before lysis and Western blotting (ii). e: The percentage of cells with nuclear FGFR following AMAXA transfection with FGFR1 plasmid and treatment with either serum free media (SF), bFGF and PDGF (GF), and 20, 100 or 200 pM Fc or SNC-Fc plus GF. * indicates a significant difference from SF control, # indicates a significant difference from GF and Fc plus GF, n = 3. Representative images to show nuclear translocation of FGFR1 for the 200 pM dose. Scale bars represent 10 mm. Yellow arrowheads indicate cells with nuclear FGFR1 (green), white arrowheads indicate cells with cytoplasmic FGFR1. Nuclei have been psuedocoloured in red. f: Proliferation (percentage of BrdU positive human VSMCs) following transfection with FGFR1(SP/NLS), a constitutively nuclear form of FGFR1, and treatment with 200 pM Fc or SNC-Fc in the presence of bFGF and PDGF for 24 h. * indicates a significant difference from SF, n = 3.

Article Snippet: Antibodies were used at the following concentrations: N-cadherin (BD Biosciences: 610920, 1:2500), GAPDH (Chemicon: MAB374, 1:5000), and FGFR1 (R&D Systems: MAB658, 1:50).

Techniques: Control, Incubation, Binding Assay, Mutagenesis, Western Blot, Immunoprecipitation, Lysis, Transfection, Plasmid Preparation, Translocation Assay

Journal: Endocrine Connections

Article Title: Intracellular FGF1 promotes invasion and migration in thyroid carcinoma via HMGA1 independent of FGF receptors

doi: 10.1530/EC-23-0014

Figure Lengend Snippet: Information for the primers used in this study.

Article Snippet: The sample extracts were then separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis at 100 V for 2 h and transferred to PVDF membranes (Millipore) at 200 mA for 2 h. Then, the PVDF membranes were blocked with 10% nonfat milk for 2 h at room temperature and incubated with the primary antibodies rabbit anti-FGF1 antibody (1:200, Abcam, WH168080), mouse anti-HMGA1 (1:2000, Santa Cruz Biotechnology, SC-393213), mouse anti-FGFR1 (1:500, Proteintech (Wuhan, China), 60325-1-lg), rabbit anti-FGFR4 (1:500, Proteintech, 11098-1-AP), rabbit anti-phospho-FGFR1 (Y653/Y654) (1:500, Abclonal, AP1317), rabbit anti-phospho-FGFR2 (Tyr463/Tyr466) (1:500, Bioss (Beijing, China), bs-5362R), rabbit anti-phospho-FGFR3 (Y724) (1:500, Abclonal, AP1274), rabbit anti-phospho-FGFR4 (Y642) (1:500, Cusabio (Wuhan, China), CSB-PA008250), mouse anti-E-cadherin (1:500, BD Biosciences (Franklin Lakes, NJ, USA), 610182), rabbit anti-vimentin (1:1000, Cell Signaling, 10366-1-AP), mouse anti-beta-actin (1:1000, ORCIGENE (Rockville, MD, USA), TAB11000), and rabbit anti-slug (1:200, Proteintech, 12129-1-AP) overnight in 5% skim milk solution at 4°C.

Techniques: Sequencing

Journal: Endocrine Connections

Article Title: Intracellular FGF1 promotes invasion and migration in thyroid carcinoma via HMGA1 independent of FGF receptors

doi: 10.1530/EC-23-0014

Figure Lengend Snippet: FGF receptors are not essential for FGF1 to promote EMT in thyroid carcinoma. (A) The mRNA expression levels of FGFR1, FGFR2, FGFR3, and FGFR4 in PTC tissues and Nor tissues. (B–C) The influences of FGF1 OE on the mRNA levels of FGFR1, FGFR2, FGFR3, and FGFR4 in B-CPAP and CAL-62 cells. (D) The influences of FGF1 OE on the phosphorylation of proteins of FGFR1, FGFR2, FGFR3, and FGFR4 in B-CPAP and CAL-62 cells. (E) The effects of FGF1 OE on the protein levels of FGFR1 and FGFR4 in B-CPAP and CAL-62 cells. (F) The effects of FGF1 OE on the protein levels of E-cadherin, Vimentin, and Slug in the presence of the FGFR1 inhibitor SU5402 in B-CPAP and CAL-62 cells. (G) The influences of FGF1 OE on the protein levels of E-cadherin, Vimentin, and Slug in the presence of the FGFR4 inhibitor H3B-6527 in B-CPAP and CAL-62 cells. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The sample extracts were then separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis at 100 V for 2 h and transferred to PVDF membranes (Millipore) at 200 mA for 2 h. Then, the PVDF membranes were blocked with 10% nonfat milk for 2 h at room temperature and incubated with the primary antibodies rabbit anti-FGF1 antibody (1:200, Abcam, WH168080), mouse anti-HMGA1 (1:2000, Santa Cruz Biotechnology, SC-393213), mouse anti-FGFR1 (1:500, Proteintech (Wuhan, China), 60325-1-lg), rabbit anti-FGFR4 (1:500, Proteintech, 11098-1-AP), rabbit anti-phospho-FGFR1 (Y653/Y654) (1:500, Abclonal, AP1317), rabbit anti-phospho-FGFR2 (Tyr463/Tyr466) (1:500, Bioss (Beijing, China), bs-5362R), rabbit anti-phospho-FGFR3 (Y724) (1:500, Abclonal, AP1274), rabbit anti-phospho-FGFR4 (Y642) (1:500, Cusabio (Wuhan, China), CSB-PA008250), mouse anti-E-cadherin (1:500, BD Biosciences (Franklin Lakes, NJ, USA), 610182), rabbit anti-vimentin (1:1000, Cell Signaling, 10366-1-AP), mouse anti-beta-actin (1:1000, ORCIGENE (Rockville, MD, USA), TAB11000), and rabbit anti-slug (1:200, Proteintech, 12129-1-AP) overnight in 5% skim milk solution at 4°C.

Techniques: Expressing

Journal: Journal of translational medicine

Article Title: A novel intronic circular RNA circFGFR1 int2 up-regulates FGFR1 by recruiting transcriptional activators P65/FUS and suppressing miR-4687-5p to promote prostate cancer progression.

doi: 10.1186/s12967-023-04718-y

Figure Lengend Snippet: Fig. 6 miR-4687-5p was downregulated in PCa and interacted with circFGFR1int2. A Bioinformatics analysis (RegRNA 2.0 and RNA22 v2) of hsa-miR-4687-5p showing the potential circFGFR1int2-interacting sequence TGTGGGGTGAGGGCT (in red) at 762 to 779. B, C miR-4687-5p was significantly down-regulated in PCa cells (LNCap, PC-3, and DU145) and tissue samples (n = 6) than in normal prostate cell RWPE-1 and BPH tissues. D Expression of FGFR1 mRNA was negatively correlated with that of miR-4687-5p. E Dot blot hybridization showed biotin-labelled miR-4687-5p probe bind to wild-type circFGFR1int2 (WT) in dose-dependent manner, whereas mutation of the binding site (MUT) resulted in no or very weak signals. Blank (no RNA was added) was used as negative control. Error bars for qRT-PCR represented mean ± standard deviation (SD) of three independent experiments. **P < 0.01, ***P < 0.001, ns, not significant

Article Snippet: Paraffin sections were boiled in 1 × EDTA for antigen retrieval, and incubated with FGFR1 antibody (mouse monoclonal, 1:200, 60,325-1, Proteintech) for 2 h at 37 °C, then with secondary antibody (PV-6000D, ZSGBBIO, Beijing, China) for 1 h at 37 °C.

Techniques: Sequencing, Expressing, Dot Blot, Hybridization, Mutagenesis, Binding Assay, Negative Control, Quantitative RT-PCR, Standard Deviation

Journal: Journal of translational medicine

Article Title: A novel intronic circular RNA circFGFR1 int2 up-regulates FGFR1 by recruiting transcriptional activators P65/FUS and suppressing miR-4687-5p to promote prostate cancer progression.

doi: 10.1186/s12967-023-04718-y

Figure Lengend Snippet: Fig. 7 miR-4687-5p was a novel suppressor of FGFR1 by targeting FGFR1 3′UTR and CDS. A Bioinformatics analysis by TargetScan, miRwalk, Microt4, miRanda and RNAhybrid databases revealed two potential miR-4687-5p-interacting sites located in FGFR1 3′UTR and CDS, respectively. B The miR-4687-5p binding sequences in 3′UTR (5′-AGGGCU-3′, red) and CDS (5′-GAGGGCTG-3′, blue) were highly conserved across species. C Artificial overexpression of miR-4687-5p by mimics significantly suppressed expression of FGFR1 mRNA and protein. D Dot blot hybridization showing biotin-labelled miR-4687-5p probe bound to wild-type FGFR1 3′UTR and CDS fragments in a dose-dependent manner, while mutation of miR-4687-5p binding sites in FGFR1 mRNA (FGFR1 3′UTR-MUT and FGFR1 CDS-MUT) resulted in no or signals. Error bars for qRT-PCR represented mean ± standard deviation (SD) of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Paraffin sections were boiled in 1 × EDTA for antigen retrieval, and incubated with FGFR1 antibody (mouse monoclonal, 1:200, 60,325-1, Proteintech) for 2 h at 37 °C, then with secondary antibody (PV-6000D, ZSGBBIO, Beijing, China) for 1 h at 37 °C.

Techniques: Binding Assay, Over Expression, Expressing, Dot Blot, Hybridization, Mutagenesis, Quantitative RT-PCR, Standard Deviation

Journal: Oncogene

Article Title: Nuclear FGFR1 promotes pancreatic stellate cell-driven invasion through up-regulation of Neuregulin 1

doi: 10.1038/s41388-022-02513-5

Figure Lengend Snippet: A Schematic of spheroid model. Cancer cells and PSCs are placed in methylcellulose hanging drops to form spheres. Spheres are subsequently placed in Collagen: Matrigel hydrogels and cultured for 3 days. B Brightfield (Top panels) and confocal (Lower panels) images of MIA PaCa-2: PS1 spheres cultured with either DMSO or 1 μM AZD4547 (FGFR inhibitor). MIA PaCa-2 cancer cells are labelled with H2B-RFP (purple) and PS1 PSCs with H2B-GFP (green). Quantification of relative spheroid invasion and central spheroid size also presented. C Schematic of organotypic cultures. Cancer cell and PSCs are cultured on top of a Collagen: Matrigel hydrogel and cultured for 7 days. D H&E images of MIA PaCa-2: PS1 organotypics cultured with either DMSO or 1 μM AZD4547. Quantification of cell invasion also presented. E Brightfield images of MIA PaCa-2: PSC25 spheres cultured with either DMSO or 1 μM AZD4547, presented with relative spheroid invasion and central spheroid area quantification. F Western blot of FGFR1 expression in PS1 cells harbouring inducible FGFR1 shRNA_a treated with or without 1 μg/mL doxycycline (Dox) for 48 h. G Brightfield (Top panels) and H&E (Lower panels) images of MIA PaCa-2: PS1 spheroids (Top Panels) and organotypics (Lower Panels) with inducible expression of either a control shRNA or FGFR1 shRNA_a in the PSCs. All images representative of at least 3 biological repeats. Individual colours on graphs indicative of technical replicates within each biological replicate. **** P < 0.0001, ** P < 0.01, NS Not Significant, Two-tailed T test. Scale bar = 100 μm.

Article Snippet: FGFR1 (9740, Cell Signalling) , Rabbit , 1:500 (WB).

Techniques: Cell Culture, Western Blot, Expressing, shRNA, Control, Two Tailed Test

Journal: Oncogene

Article Title: Nuclear FGFR1 promotes pancreatic stellate cell-driven invasion through up-regulation of Neuregulin 1

doi: 10.1038/s41388-022-02513-5

Figure Lengend Snippet: A Representative confocal images of FGFR1 (white) and Vimentin (green) in COLO 357: PS1 organotypic cross sections. B Representative immunofluorescence confocal image of FGFR1 expression (white) in MIA PaCa-2: PS1 spheroids. Cancer cells labelled with H2B-RFP (purple), PSCs labelled with H2B-GFP (green). C Brightfield images of MIA PaCa-2: PSC25 spheres with PS1-specific knockdown of Importin β. Quantification of relative spheroid invasion and relative central sphere size presented to right of image panels. Individual colours on graphs indicative of technical replicates within each biological replicate. D Schematic of FGFR1 ChIP-Seq. E Pie chart of FGFR1-DNA binding regions identified within the genome in PS1 cells. F Pathway enrichment of FGFR1-DNA binding regions identified in PS1 cells. G Heat-map of FGFR1-DNA binding peaks taken from ChIP-Seq data of an individual replicate of PS1 cells treated with either DMSO or 1 μM AZD4547 for 24 h. **** P < 0.0001, Two-tailed T test. Scale bar = 100 μm, =20 μm for insets.

Article Snippet: FGFR1 (9740, Cell Signalling) , Rabbit , 1:500 (WB).

Techniques: Immunofluorescence, Expressing, Knockdown, ChIP-sequencing, Binding Assay, Two Tailed Test

Journal: Oncogene

Article Title: Nuclear FGFR1 promotes pancreatic stellate cell-driven invasion through up-regulation of Neuregulin 1

doi: 10.1038/s41388-022-02513-5

Figure Lengend Snippet: A Integrative Genomics Viewer (IGV) snapshot of peaks of FGFR1-DNA binding identified within NRG1 gene (green box) with and without treatment with 1 μM AZD4547 (FGFR inhibitor). B ChIP-PCR of FGFR1 binding to NRG1 in PS1 cells treated with DMSO or 1 μM AZD4547 for 24 h. C NRG1 expression in PS1 cells treated with DMSO or 1 μM AZD4547 for 24 h. D Brightfield images of MIA PaCa-2: PS1 (Top panels) and MIA PaCa-2: mPSC (Lower panels) spheres treated with either recombinant NRG1 (100 ng/mL) or 1 μM AZD4547 for 3 days either alone or in combination. Quantification of relative spheroid invasion presented below image panels ( D’ , D” ). E Brightfield images of MIA PaCa-2: PS1 spheres with either MIA PaCa-2 (Top panels) or PS1 (Lower panels) knockdown of indicated ERBB gene. Quantification of relative spheroid invasion presented next to image panels ( E’ , E” ). F Schematic of proposed interaction between FGFR1 and NRG1 in PSCs. FGFR1 translocates to the nucleus where it induces expression of NRG1 . NRG1 then signals back on PSCs through an ERBB2/4 heterodimer to promote invasion. All images representative of at least 3 biological repeats. Individual colours on graphs indicative of technical replicates within each biological replicate. **** P < 0.0001, *** P < 0.001, * P < 0.05, NS Not Significant, Two-tailed T test or ANOVA with Dunnett’s post hoc test. Scale bar = 100 μm.

Article Snippet: FGFR1 (9740, Cell Signalling) , Rabbit , 1:500 (WB).

Techniques: Binding Assay, Expressing, Recombinant, Knockdown, Two Tailed Test

Journal: Oncogene

Article Title: Nuclear FGFR1 promotes pancreatic stellate cell-driven invasion through up-regulation of Neuregulin 1

doi: 10.1038/s41388-022-02513-5

Figure Lengend Snippet: Antibody conditions.

Article Snippet: FGFR1 (9740, Cell Signalling) , Rabbit , 1:500 (WB).

Techniques: