Journal: Nature Communications

Article Title: Sensory nerve niche regulates mesenchymal stem cell homeostasis via FGF/mTOR/autophagy axis

doi: 10.1038/s41467-023-35977-4

Figure Lengend Snippet: a Expression of FGF1 and β-gal in one-month-old Gli1-LacZ mice. b Relative contributions of ligand-receptor pairs to the overall communication network of FGF signaling pathway; the Fgf1-Fgfr1 pair is the major contributor. c Expression of FGFR1 and β-gal in one-month-old Gli1-LacZ mice. d Immunoprecipitation assay demonstrating the interaction between FGF1 and FGFR1 in the proximal mesenchyme of the incisor. e Expression of Fgfr1 and tdTomato in MSC cultures from Gli1 CreER ;tdTomato mice. f – j Abnormal dentin formation in Gli1 CreER ;Fgfr1 fl/fl mice three months after tamoxifen induction. f CT imaging of the incisors of Fgfr1 fl/fl and Gli1 CreER ;Fgfr1 fl/fl mice. White arrow points to the dental pulp cavity; the white arrowhead points to narrowed pulp cavity. g Histological analysis of Fgfr1 fl/fl and Gli1 CreER ;Fgfr1 fl/fl mice. Yellow arrow points to pre-odontoblast; yellow arrowhead points to abnormal pre-odontoblast; asterisk points to abnormal dentin formation. h Expression of Dspp in control and Gli1 CreER ;Fgfr1 fl/fl mice. Yellow arrow points to the distance between the bending point of the cervical loop and the initiation of odontoblast differentiation. i Quantification of dental pulp cavity percentage in control and mutant mice. P = 0.0008. j Quantification of the distance of Dspp + cells to cervical loop in control and mutant mice. P < 0.0001. Schematic at the bottom indicates the induction protocol. mpt month post-tamoxifen injection. k The growth rate of the incisor was detected with notch movement observed at day (D)3, D6, and D14 in control and Gli1 CreER ;Fgfr1 fl/fl mice. White arrow points to the notch location; the schematic at the bottom indicates the induction protocol. mpt month post-tamoxifen injection. l Quantification of notch movement in control and mutant mice. P = 0.0123. For I , j and l , n = 3 and each data point represent one animal, with unpaired Student’s t -test performed. All data are expressed as the mean ± SD. Source data are provided as a Source Data file. *** P < 0.001, **** P < 0.0001. Each experiment was repeated independently three times. White dotted line outlines the cervical loop. Cre-: Fgfr1 fl/fl ; Cre+: Gli1 CreER ;Fgfr1 fl/fl . Scale bars, f 1 mm; k 2 mm; e 50 μm; others, 100 μm.

Article Snippet: Membranes were blocked with 5% non-fat dry milk for 1 h, then incubated with primary antibodies: anti-FGFR1 (Cell signaling technology 9740, 1:1000), anti-FGF1 (Abcam ab207321, 1:1000), anti-LC3 (1:1000, Abcam, ab48394), anti-p-ULK1 (Cell Signaling technology 14202, 1:1000), anti-p-mTOR (Cell Signaling technology 5536, 1:1000), anti-mTOR (Cell Signaling technology 2972, 1:1000), anti-p-p70S6K (Cell Signaling technology 9204, 1:1000), anti-p70S6K (Cell Signaling technology 2708, 1:1000), anti-p-S6 (Cell Signaling technology 4858, 1:1000), anti-S6 (Cell Signaling technology 2217, 1:1000), anti-p-Akt (Cell Signaling technology 4060, 1:1000), anti-Akt (Cell Signaling technology 9272, 1:1000), anti-p-p38 (Cell Signaling technology 4511, 1:1000), anti-p38 (Cell Signaling technology 8690, 1:1000), anti-p-ERK (Cell Signaling technology 4370, 1:1000), anti-ERK (Cell Signaling technology 4695, 1:1000), anti-p-JNK (Cell Signaling technology 9255, 1:1000), anti-JNK (Cell Signaling technology 9252, 1:1000), and anti-β-actin (Abcam ab20272, 1:1000) at 4 °C overnight.

Techniques: Expressing, Immunoprecipitation, Imaging, Mutagenesis, Injection

Journal: Nature Communications

Article Title: Sensory nerve niche regulates mesenchymal stem cell homeostasis via FGF/mTOR/autophagy axis

doi: 10.1038/s41467-023-35977-4

Figure Lengend Snippet: a–d The number of MSCs decreased in Gli1 CreER ;Fgfr1 fl/fl mice one week after tamoxifen induction. Gli1 + cells stained with β-gal in incisors of control and Gli1 CreER ;Fgfr1 fl/fl mice. e , Quantification of the percentage of Gli1 + cells in control and mutant mice. P = 0.0007. f–i TACs detected with Ki67 staining in control and Gli1 CreER ;Fgfr1 fl/fl mice. j Quantification of Ki67 + TACs in control and mutant mice. P = 0.0008. k–n The expression of Dspp and EdU in control and Fgfr1 fl/fl mutant mice. The length of overlap between Dspp + odontoblasts and EdU + cells reflects the number of TACs undergoing odontoblastic differentiation. White arrowhead points to overlap between Dspp + odontoblasts and EdU + cells. o Quantification of overlap between Dspp + odontoblasts and EdU + cells in control and mutant mice. P = 0.0011. p and q The migration of Gli1 + cells’ progeny indicated with tdTomato in Gli1 CreER ;tdTomato and Gli1 CreER ;Fgfr1 fl/fl ; tdTomato mice. White arrows point to tdTomato + cell migration. Yellow dot line with arrowheads point to migration distance. r Quantification of the percentage of tdTomato length in control and mutant mice. P = 0.0005. For e, o and r , n = 3 and each data point represents one animal, with unpaired Student’s t -test performed. All data are expressed as the mean ± SD. Source data are provided as a Source Data file. ** P < 0.01, *** P < 0.001. Each experiment was repeated independently three times. White dotted line outlines the cervical loop. Scale bars, p and q 400 μm; others, 100 μm.

Article Snippet: Membranes were blocked with 5% non-fat dry milk for 1 h, then incubated with primary antibodies: anti-FGFR1 (Cell signaling technology 9740, 1:1000), anti-FGF1 (Abcam ab207321, 1:1000), anti-LC3 (1:1000, Abcam, ab48394), anti-p-ULK1 (Cell Signaling technology 14202, 1:1000), anti-p-mTOR (Cell Signaling technology 5536, 1:1000), anti-mTOR (Cell Signaling technology 2972, 1:1000), anti-p-p70S6K (Cell Signaling technology 9204, 1:1000), anti-p70S6K (Cell Signaling technology 2708, 1:1000), anti-p-S6 (Cell Signaling technology 4858, 1:1000), anti-S6 (Cell Signaling technology 2217, 1:1000), anti-p-Akt (Cell Signaling technology 4060, 1:1000), anti-Akt (Cell Signaling technology 9272, 1:1000), anti-p-p38 (Cell Signaling technology 4511, 1:1000), anti-p38 (Cell Signaling technology 8690, 1:1000), anti-p-ERK (Cell Signaling technology 4370, 1:1000), anti-ERK (Cell Signaling technology 4695, 1:1000), anti-p-JNK (Cell Signaling technology 9255, 1:1000), anti-JNK (Cell Signaling technology 9252, 1:1000), and anti-β-actin (Abcam ab20272, 1:1000) at 4 °C overnight.

Techniques: Staining, Mutagenesis, Expressing, Migration

Journal: Nature Communications

Article Title: Sensory nerve niche regulates mesenchymal stem cell homeostasis via FGF/mTOR/autophagy axis

doi: 10.1038/s41467-023-35977-4

Figure Lengend Snippet: a–i mTOR-dependent autophagy is downregulated in Gli1 CreER ;Fgfr1 fl/fl mice. a Hierarchical clustering of control and Gli1 CreER ;Fgfr1 fl/fl mice. b Volcano plot showing 2019 upregulated genes and 1447 downregulated genes. c Significant signaling pathways analyzed with Ingenuity Pathway Analysis (IPA). d Expression of FGFR1, LC3 and p-ULK1 in mesenchyme of incisors from control and Gli1 CreER ;Fgfr1 fl/fl mice. e–h Expression of autophagy substrate P62 in control and Gli1 CreER ;Fgfr1 fl/fl mice. i Expression of p-mTOR and its downstream effectors p-P70S6K and pS6 in control and mutant mice. j–p FGF/p-JNK signaling regulates mTOR-dependent autophagy activation in MSCs. j Western blot of p-AKT, AKT, p-P38, P38, p-ERK, ERK, p-JNK, and JNK in proximal mesenchyme from control and mutant mice. k and l Expression of p-JNK and β-gal in the proximal mesenchyme of incisors from Gli1-LacZ mice. m–p Expression of p-JNK in control and Gli1 CreER ;Fgfr1 fl/fl mice. q Relative protein level in ( d ). FGFR1/β-Actin: P = 0.0001; LC3II/LC3I: P = 0.0006; p-ULK1/β-Actin: P < 0.0001. r Relative protein level in ( i ). p-mTOR/mTOR: P = 0.0056; p-P70S6K/P70S6K: P = 0.0016; pS6/S6: P = 0.0001. s Relative protein level in ( j ). p-Akt/Akt: P = 0.3485; p-P38/P38: P = 0.9988; p-ERK/ERK: P = 0.0025; p-JNK/JNK: P < 0.0001. t Relative fluorescent intensity of P62 and p-JNK. P62: P < 0.0001; p-JNK: P = 0.0019. For q–t , n = 3 and each data point represent one biological replicate, with unpaired Student’s t -test performed. All data are expressed as the mean ± SD. Source data are provided as a Source Data file. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Each experiment was repeated independently three times. White dotted line outlines the cervical loop. Scale bars, 100 μm.

Article Snippet: Membranes were blocked with 5% non-fat dry milk for 1 h, then incubated with primary antibodies: anti-FGFR1 (Cell signaling technology 9740, 1:1000), anti-FGF1 (Abcam ab207321, 1:1000), anti-LC3 (1:1000, Abcam, ab48394), anti-p-ULK1 (Cell Signaling technology 14202, 1:1000), anti-p-mTOR (Cell Signaling technology 5536, 1:1000), anti-mTOR (Cell Signaling technology 2972, 1:1000), anti-p-p70S6K (Cell Signaling technology 9204, 1:1000), anti-p70S6K (Cell Signaling technology 2708, 1:1000), anti-p-S6 (Cell Signaling technology 4858, 1:1000), anti-S6 (Cell Signaling technology 2217, 1:1000), anti-p-Akt (Cell Signaling technology 4060, 1:1000), anti-Akt (Cell Signaling technology 9272, 1:1000), anti-p-p38 (Cell Signaling technology 4511, 1:1000), anti-p38 (Cell Signaling technology 8690, 1:1000), anti-p-ERK (Cell Signaling technology 4370, 1:1000), anti-ERK (Cell Signaling technology 4695, 1:1000), anti-p-JNK (Cell Signaling technology 9255, 1:1000), anti-JNK (Cell Signaling technology 9252, 1:1000), and anti-β-actin (Abcam ab20272, 1:1000) at 4 °C overnight.

Techniques: Expressing, Mutagenesis, Activation Assay, Western Blot

Journal: Nature Communications

Article Title: Sensory nerve niche regulates mesenchymal stem cell homeostasis via FGF/mTOR/autophagy axis

doi: 10.1038/s41467-023-35977-4

Figure Lengend Snippet: a–e Abnormal dentin deposition seen in Gli1 CreER ;Fgfr1 fl/fl mice can be rescued by rapamycin treatment for 3 months. a CT scanning of control, Gli1 CreER ;Fgfr1 fl/fl mice treated with vehicle ( Gli1 CreER ;Fgfr1 fl/fl + Veh) and Gli1 CreER ;Fgfr1 fl/fl mice treated with rapamycin ( Gli1 CreER ;Fgfr1 fl/fl + Rap). White arrow points to the dental pulp cavity; white arrowhead points to narrowed pulp cavity. b Histological analysis of these three groups. Yellow arrow points to normal pre-odontoblast; yellow arrowhead points to abnormal pre-odontoblast; asterisk points to abnormal dentin formation. c Expression of Dspp in these three groups. Yellow arrow points to the distance between the bending point of the cervical loop and the initiation of odontoblast differentiation. d Quantification of dental pulp cavity percentage in these three groups. Control vs Gli1 CreER ;Fgfr1 fl/fl + Veh: P = 0.0001; Gli1 CreER ;Fgfr1 fl/fl + Veh vs. Gli1 CreER ;Fgfr1 fl/fl + Rap: P = 0.0006. e Quantification of the distance of Dspp + cells to cervical loop. Control versus Gli1 CreER ;Fgfr1 fl/fl + Veh: P < 0.0001; Gli1 CreER ;Fgfr1 fl/fl + Veh versus Gli1 CreER ;Fgfr1 fl/fl + Rap: P < 0.0001. f Re-activation of autophagy benefits the retention of MSCs. Gli1 + cells labeled with β-gal in control, Gli1 CreER ;Fgfr1 fl/fl mice treated with vehicle or rapamycin. g Quantification of the percentage of Gli1 + cells. Control versus Gli1 CreER ;Fgfr1 fl/fl + Veh: P = 0.0002; Gli1 CreER ;Fgfr1 fl/fl + Veh versus Gli1 CreER ;Fgfr1 fl/fl + Rap: P = 0.0002. h TACs detected with Ki67 staining in control, Gli1 CreER ;Fgfr1 fl/fl mice treated with vehicle or rapamycin. i Quantification of Ki67 + TAC cells. Control versus Gli1 CreER ;Fgfr1 fl/fl + Veh: P < 0.0001; Gli1 CreER ;Fgfr1 fl/fl + Veh versus Gli1 CreER ;Fgfr1 fl/fl + Rap: P = 0.0021. For d , e , g , and I , n = 3 biologically independent samples, each data point represents one animal, with unpaired one-way ANOVA analysis. All data are expressed as the mean ± SD. Source data are provided as a Source Data file. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Each experiment was repeated independently three times. White dotted line outlines the cervical loop. Scale bars, 100 μm.

Article Snippet: Membranes were blocked with 5% non-fat dry milk for 1 h, then incubated with primary antibodies: anti-FGFR1 (Cell signaling technology 9740, 1:1000), anti-FGF1 (Abcam ab207321, 1:1000), anti-LC3 (1:1000, Abcam, ab48394), anti-p-ULK1 (Cell Signaling technology 14202, 1:1000), anti-p-mTOR (Cell Signaling technology 5536, 1:1000), anti-mTOR (Cell Signaling technology 2972, 1:1000), anti-p-p70S6K (Cell Signaling technology 9204, 1:1000), anti-p70S6K (Cell Signaling technology 2708, 1:1000), anti-p-S6 (Cell Signaling technology 4858, 1:1000), anti-S6 (Cell Signaling technology 2217, 1:1000), anti-p-Akt (Cell Signaling technology 4060, 1:1000), anti-Akt (Cell Signaling technology 9272, 1:1000), anti-p-p38 (Cell Signaling technology 4511, 1:1000), anti-p38 (Cell Signaling technology 8690, 1:1000), anti-p-ERK (Cell Signaling technology 4370, 1:1000), anti-ERK (Cell Signaling technology 4695, 1:1000), anti-p-JNK (Cell Signaling technology 9255, 1:1000), anti-JNK (Cell Signaling technology 9252, 1:1000), and anti-β-actin (Abcam ab20272, 1:1000) at 4 °C overnight.

Techniques: Expressing, Activation Assay, Labeling, Staining

Journal: Nature Communications

Article Title: Sensory nerve niche regulates mesenchymal stem cell homeostasis via FGF/mTOR/autophagy axis

doi: 10.1038/s41467-023-35977-4

Figure Lengend Snippet: Sensory nerve predominates in innervating the mouse incisor, which confers the mouse incisor as an ideal model for the study of the relationship between sensory nerves and MSCs. FGF1, a ligand secreted by sensory nerves, is enriched in the proximal mesenchymal end of the incisor and surrounding Gli1 + cell. FGF1 directly regulates MSCs in the incisor by binding to FGFR1 specifically and activates the FGF/p-JNK/mTOR/autophagy axis to regulate MSCs in maintaining tissue homeostasis.

Article Snippet: Membranes were blocked with 5% non-fat dry milk for 1 h, then incubated with primary antibodies: anti-FGFR1 (Cell signaling technology 9740, 1:1000), anti-FGF1 (Abcam ab207321, 1:1000), anti-LC3 (1:1000, Abcam, ab48394), anti-p-ULK1 (Cell Signaling technology 14202, 1:1000), anti-p-mTOR (Cell Signaling technology 5536, 1:1000), anti-mTOR (Cell Signaling technology 2972, 1:1000), anti-p-p70S6K (Cell Signaling technology 9204, 1:1000), anti-p70S6K (Cell Signaling technology 2708, 1:1000), anti-p-S6 (Cell Signaling technology 4858, 1:1000), anti-S6 (Cell Signaling technology 2217, 1:1000), anti-p-Akt (Cell Signaling technology 4060, 1:1000), anti-Akt (Cell Signaling technology 9272, 1:1000), anti-p-p38 (Cell Signaling technology 4511, 1:1000), anti-p38 (Cell Signaling technology 8690, 1:1000), anti-p-ERK (Cell Signaling technology 4370, 1:1000), anti-ERK (Cell Signaling technology 4695, 1:1000), anti-p-JNK (Cell Signaling technology 9255, 1:1000), anti-JNK (Cell Signaling technology 9252, 1:1000), and anti-β-actin (Abcam ab20272, 1:1000) at 4 °C overnight.

Techniques: Binding Assay

Journal: Translational Lung Cancer Research

Article Title: Genome-wide epigenetic and mRNA-expression profiling followed by CRISPR/Cas9-mediated gene-disruptions corroborate the MIR141/MIR200C -ZEB1/ZEB2-FGFR1 axis in acquired EMT-associated EGFR TKI-resistance in NSCLC cells

doi: 10.21037/tlcr-22-507

Figure Lengend Snippet: Contribute of FGFR1 to EMT-E-TKI-R in HCC827 cells. (A) Distribution of FC (Log2) in mRNA expression following EMT-E-TKI-R of 56 receptor kinase genes for which RNA-sequencing data were available for HCC827 cells with EMT-E-TKI-R (HCC827EMT) relative to parental HCC827 cells (HCC827PAR). Selected receptor kinase genes are highlighted. (B) Distribution of H3K36me3 and DNA-methylation at the FGFR1 locus. For H3K36me3 is shown ChIP-sequencing enrichment values from HCC827PARand HCC827EMT. Asterisks show positions with significant difference. For DNA-methylation is shown the difference in beta-values in HCC827EMT relative to HCC827PAR cells. DMRs are indicated. (C) RT-qPCR-based mRNA expression analysis of HCC827Cas9 cells harboring control sgRNA C or FGFR1 sgRNAs F1 and F3. P0 indicates cells grown in absence of erlotinib and P2 indicates that cells were grown in presence of erlotinib for two passages. Values are normalized to expression of ACTB and subsequently normalized to the expression at P0 for sgRNA C given the value 1. In all panels SD represents one sample analyzed in technical triplicates, and * indicate changes for the given passage relative to HCC827Cas9 harboring sgRNA C with P<0.05 and FC >2. (D) Colorimetric MTS-assays showing the impact of FGFR1 depletion for cell viability using increasing concentrations of erlotinib for 72 h. Left panel shows result for FGFR1 sgRNA F1 and control sgRNA C and right panel shows result for FGFR1 sgRNA F3 and control sgRNA C. Each graph represents two independent MTS assays in where each sample was examined in technical duplicates and with SDs shown. * indicate differences for given concentrations of erlotinib with P<0.05. EMT-E-TKI-R, epithelial-mesenchymal-transition-associated EGFR tyrosine-kinase-inhibitor resistance; FC, fold change; EGFR, epidermal growth factor receptor; DMR, differential methylated region; SD, standard deviation; RT-qPCR, quantitative reverse transcription polymerase chain reaction.

Article Snippet: For western blot analysis of CRISPR/Cas9-mediated depletions the following antibodies were used: rabbit anti-ZEB1 (diluted 1/750) (Bethyl Laboratories, Montgomery, TX, USA, Cat# A301-922A, RRID:AB_1524126), rabbit anti-FGFR1 (diluted 1/500) (Cell Signaling Technology, Danvers, MA, USA, Cat# 9740, RRID:AB_11178519), rabbit anti-histone-H3 (diluted 1/10,000) (Abcam Cat# ab1791, RRID:AB_302613), mouse anti-βActin (diluted 1/5,000) (Sigma-Aldrich Cat# A5316, RRID:AB_476743), goat anti-mouse HRP conjugated (diluted 1/10,000) (Agilent, Santa Clara, CA, USA, Cat# P0447, RRID:AB_2617137), and goat anti-rabbit HRP conjugated (diluted 1/10,000) (Agilent Cat# P0448, RRID:AB_2617138).

Techniques: Expressing, RNA Sequencing Assay, DNA Methylation Assay, ChIP-sequencing, Quantitative RT-PCR, Methylation, Standard Deviation, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Cancer

Article Title: Tumor-associated inflammatory microenvironment in non-small cell lung cancer: correlation with FGFR1 and TLR4 expression via PI3K/Akt pathway

doi: 10.7150/jca.26277

Figure Lengend Snippet: Correlations between of FGFR1 and TLR4 expression and clinicopathologic features.

Article Snippet: Then, after antigen retrieval, the tissue sections were incubated with FGFR1 antibodies (monoclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA), TLR4 antibodies (polyclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA) at 37°C for 1 h, and then at 4°C overnight.

Techniques: Expressing, Histopathology

Journal: Journal of Cancer

Article Title: Tumor-associated inflammatory microenvironment in non-small cell lung cancer: correlation with FGFR1 and TLR4 expression via PI3K/Akt pathway

doi: 10.7150/jca.26277

Figure Lengend Snippet: Expression of FGFR1 in cancer tissues and paracancerous tissues.

Article Snippet: Then, after antigen retrieval, the tissue sections were incubated with FGFR1 antibodies (monoclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA), TLR4 antibodies (polyclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA) at 37°C for 1 h, and then at 4°C overnight.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Tumor-associated inflammatory microenvironment in non-small cell lung cancer: correlation with FGFR1 and TLR4 expression via PI3K/Akt pathway

doi: 10.7150/jca.26277

Figure Lengend Snippet: Immunohistochemical staining of FGFR1 and TLR4 in NSCLC tissues. No staining was detected for (A) FGFR1 and (B) TLR4 in the control group. Staining of FGFR1 (C) and TLR4 (D) in adenocarcinoma cells. Staining of FGFR1 (E) and TLR4 (F) in squamous carcinoma cells. (All photos are shown at 100 magnification).

Article Snippet: Then, after antigen retrieval, the tissue sections were incubated with FGFR1 antibodies (monoclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA), TLR4 antibodies (polyclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA) at 37°C for 1 h, and then at 4°C overnight.

Techniques: Immunohistochemical staining, Staining

Journal: Journal of Cancer

Article Title: Tumor-associated inflammatory microenvironment in non-small cell lung cancer: correlation with FGFR1 and TLR4 expression via PI3K/Akt pathway

doi: 10.7150/jca.26277

Figure Lengend Snippet: The relationship between FGFR1 and TLR4 expression in NSCLC.

Article Snippet: Then, after antigen retrieval, the tissue sections were incubated with FGFR1 antibodies (monoclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA), TLR4 antibodies (polyclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA) at 37°C for 1 h, and then at 4°C overnight.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Tumor-associated inflammatory microenvironment in non-small cell lung cancer: correlation with FGFR1 and TLR4 expression via PI3K/Akt pathway

doi: 10.7150/jca.26277

Figure Lengend Snippet: PI3K/Akt signaling is one of common pathway of the FGFR1 and TLR4 activation in NSCLC cells. A: The cells were respectively treated with culture medium (control group), TAK-242 control group, and 1ug/ml LPS for 24 hours, LPS with TAK-242 group. The expression of phosphorylated Akt was measured by Western Blot, * p < 0.05 vs the control group; ** p < 0.01 vs the control group; # p < 0.05 vs the LPS group; ## p < 0.01 vs the LPS group.B: The cells were respectively treated with culture medium (control group), BIBF1120 group, and 10ng/ml bFGF for 24 hours, bFGF with BIBF1120 group. The expression of phosphorylated Akt was measured by Western Blot, * p < 0.05 vs the control group; ** p < 0.01 vs the control group; # p < 0.05 vs the bFGF group; ## p < 0.01 vs the bFGF group.

Article Snippet: Then, after antigen retrieval, the tissue sections were incubated with FGFR1 antibodies (monoclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA), TLR4 antibodies (polyclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA) at 37°C for 1 h, and then at 4°C overnight.

Techniques: Activation Assay, Expressing, Western Blot

Journal: Journal of Cancer

Article Title: Tumor-associated inflammatory microenvironment in non-small cell lung cancer: correlation with FGFR1 and TLR4 expression via PI3K/Akt pathway

doi: 10.7150/jca.26277

Figure Lengend Snippet: PI3K/Akt signaling is involved in release of TNF-α and IL-6 induced by the FGFR1 and TLR4 agonists. The A549, PC-9 and SK-MES-1 (n=3) cells were respectively treated with culture medium, 1ug/ml LPS or 10ng/ml bFGF, 10µM LY294002 with or without LPS or bFGF. A:the expression of TNF-α was measured by ELISA(Mean ± SD). B: the expression of IL-6 was measured by ELISA (Mean ± SD). * p < 0.05 vs the control group, ** p < 0.01 vs the control group, # p < 0.05 vs the LPS group, ## p < 0.01 vs the LPS group, & p < 0.05 vs the bFGF group, && p < 0.01 vs the bFGF group.

Article Snippet: Then, after antigen retrieval, the tissue sections were incubated with FGFR1 antibodies (monoclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA), TLR4 antibodies (polyclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA) at 37°C for 1 h, and then at 4°C overnight.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cancer

Article Title: Tumor-associated inflammatory microenvironment in non-small cell lung cancer: correlation with FGFR1 and TLR4 expression via PI3K/Akt pathway

doi: 10.7150/jca.26277

Figure Lengend Snippet: FGFR1 regulated cell proliferation and migration by the PI3K/Akt pathway. The PC-9 cells were divided into control group, LY294002 group, bFGF group and LY294002 with bFGF group. A: the cell proliferation assay was measured by xCELLigence RTCA system. B: the the cell migration assay was measured by xCELLigence RTCA system.

Article Snippet: Then, after antigen retrieval, the tissue sections were incubated with FGFR1 antibodies (monoclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA), TLR4 antibodies (polyclonal rabbit anti-human, 1:500, Cell Signaling Technology Co., Ltd, MA, USA) at 37°C for 1 h, and then at 4°C overnight.

Techniques: Migration, Proliferation Assay, Cell Migration Assay

Journal: BioMed Research International

Article Title: Isolation and Characterization of Human Lung Lymphatic Endothelial Cells

doi: 10.1155/2015/747864

Figure Lengend Snippet: Detection of Receptor Tyrosine Kinases on HL-LECs (P4). (a) and (b) (immunofluorescence): green fluorescence corresponds to the surface expression of vascular endothelial growth factor receptor- (VEGFR-) 2 and VEGFR-3, respectively, in cultured HL-LECs. (c): representative image of a negative control in which the primary antibody was omitted from the reaction. Nuclei are shown by the blue fluorescence of DAPI. (d)–(j) (immunoperoxidase): the brownish precipitate on the surface of HL-LECs on each panel corresponds respectively to the expression of Platelet-Derived Growth Factor Receptor- (PDGFR-) beta, Epidermal Growth Factor Receptor- (EGFR-) 1, Fibroblast Growth Factor Receptor- (FGFR-) 1, type 1 Insulin-like Growth Factor Receptor (IGF-1R), the Hepatocyte Growth Factor receptor c-MET, Tropomyosin-related kinases A (TrkA), and neurotrophin p75 receptor (p75 NTR ). (k): representative image of a negative control in which the primary antibody was omitted from the reaction. Nuclei are counterstained with Haematoxylin. Scale bars: (a), (d), (e), (f), (g), (h), (i), (j), and (k) = 50 μ m; (b) and (c) = 250 μ m.

Article Snippet: Briefly, after fixation, chamber slides of HL-LECs were immersed in 3% hydrogen peroxide solution for 10 minutes and then incubated with mouse anti-human D2-40 (1 : 50; o/n 4°C; Biocare Medical, Concord, CA, USA), rabbit anti-human LYVE-1 (1 : 50; o/n 4°C; Abcam, Cambridge, UK), rabbit anti-human FGFR-1 (1 : 40; o/n 4°C; Cell Signaling, Beverly, MA, USA), mouse anti-human EGFR (1 : 40; o/n 4°C; Zymed-Invitrogen, Grand Island, NY, USA), rabbit anti-human c-MET (according to manufacturer's recommendations; Ventana-Roche), rabbit anti-human IGF-1R (1 : 30; o/n 4°C; Cell Signaling), rabbit anti-human PDGFR-beta (1 : 25; o/n 4°C; Abcam), rabbit anti-human TrkA (1 : 40; o/n 4°C; Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit anti-human p75 NTR (1 : 70; o/n 4°C; Millipore, Darmstadt, Germany), respectively.

Techniques: Immunofluorescence, Fluorescence, Expressing, Cell Culture, Negative Control, Derivative Assay

Journal: Theranostics

Article Title: FGFR blockade boosts T cell infiltration into triple-negative breast cancer by regulating cancer-associated fibroblasts

doi: 10.7150/thno.68972

Figure Lengend Snippet: FGFR signaling pathways were enriched in immune-excluded type triple-negative breast cancer (TNBC). A) The gene signature in immune-inflamed and immune-excluded TNBC samples from TCGA dataset. B) The correlation between FGFR score and 23 types of stromal cells in TME based on TCGA BRCA dataset. “Pos.” represents immune cells positively correlated with FGFR score; “Neg.” represents immune cells negatively correlated with FGFR score; “No correlation” represents immune cells that do not correlate with FGFR score. Immune subtypes (C1-C6) were characterized by differences in the nature of the overall immune response .C) The correlation between FGFR1/2/3/4 expression and cytotoxic T lymphocytes (CTL) infiltration in indicated cancer types from GEO database based on Tumor Immune Dysfunction and Exclusion (TIDE) system. D) T cell exclusion score in BRCA of TCGA based on FGFRs expression. E) Immune phenotypes of TNBC defined by IHC staining of CD3. F) The expression of FGFR1 in immune-inflamed and immune-excluded TNBC samples based on IHC staining (inflamed, n=33; excluded, n=118, t test). G) Kaplan-Meier survival analysis of low FGFR1 (blue, n=51) versus high FGFR1 (red, n=68) expression in TNBC.

Article Snippet: Tumor specimens were stained using antibodies against α-SMA (Abcam, ab7817), CD3 (Abcam, ab5690) or FGFR1 (Cell Signaling Technology, #9740).

Techniques: Expressing, Immunohistochemistry

Journal: Theranostics

Article Title: FGFR blockade boosts T cell infiltration into triple-negative breast cancer by regulating cancer-associated fibroblasts

doi: 10.7150/thno.68972

Figure Lengend Snippet: FGFR blockade induced T cell infiltration by modulating fibroblasts. A) The effect of FGFRi Erdafitinib on CD4 + and CD8 + T cell migration was detected by transwell migration assay (n=5 biological replicates, one-way ANOVA). B) Representative staining and IHC score of α-SMA in immune-inflamed and immune-excluded TNBC samples. C) Representative IF staining of α-SMA and CD3 in immune-inflamed and immune-excluded TNBC samples. D) FGFR1 expression in tumor microenvironment of breast cancer (GSE114727). E-F) Cell population in TME of breast cancer based on FGFR1 expression. G) Representative IF staining of FGFR1 and α-SMA in TNBC samples. H) The effect of FGFRi Erdafitinib on CD4 + and CD8 + T cell migration in presence of CAFs was detected by transwell migration assay (n=3 biological replicates, one-way ANOVA).

Article Snippet: Tumor specimens were stained using antibodies against α-SMA (Abcam, ab7817), CD3 (Abcam, ab5690) or FGFR1 (Cell Signaling Technology, #9740).

Techniques: Migration, Transwell Migration Assay, Staining, Expressing

Journal: Theranostics

Article Title: FGFR blockade boosts T cell infiltration into triple-negative breast cancer by regulating cancer-associated fibroblasts

doi: 10.7150/thno.68972

Figure Lengend Snippet: FGFR blockade synergizes with immune checkpoint blockade therapy. A) Overall survival of melanoma patients who had high FGFR1 vs. low FGFR1 expressed in the tumors before anti-PD-1 treatment (GSE78220). B and C) EMT6 (B) and 4T1 (C) tumor growth in mice treated with vehicle, anti-PD-1, FGFRi (Erdafitinib) or combination of anti-PD-1 and FGFRi (n=7 mice/group, two-way ANOVA). D) Survival analysis of 4T1 tumor-bearing mice treated with indicated therapy (n=8 mice/group, log-rank test). E) The t-SNE plot of TILs and CD8 + T cell population in 4T1 tumors from mice treated with indicated therapies (n=6, one-way ANOVA). F) Percentage of IFN-γ + CD8 + T-cells in indicated therapy-treated 4T1 tumors (n=6, one-way ANOVA). G) Gene ontology (GO) analysis by RNA-seq of 4T1 tumors in indicated groups (n=3/group). Heatmap shows the DEGs and associated signatures. COM, anti-PD-1+FGFRi. H) Heatmap shows the percentage of tumor infiltrating immune cells and fibroblasts in indicated therapy-treated 4T1 tumors.

Article Snippet: Tumor specimens were stained using antibodies against α-SMA (Abcam, ab7817), CD3 (Abcam, ab5690) or FGFR1 (Cell Signaling Technology, #9740).

Techniques: RNA Sequencing Assay

Journal: Theranostics

Article Title: Peptide ligands targeting FGF receptors promote recovery from dorsal root crush injury via AKT/mTOR signaling

doi: 10.7150/thno.62525

Figure Lengend Snippet: Role of FGFR signaling in response to the CH02 peptide on promoting nerve regeneration. (A) Western blot analysis of phosphorylated FGFR expression in C5-T1 DRGs subjected to dorsal root crush injury, followed by CH02 treatment (20 μM). DRGs were collected after 3 days. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were used as the loading control. (B) Western blot analysis of phosphorylated FGFR expression in ex vivo cultured DRG explants of adult Sprague-Dawley rats in response to CH02 treatment at different induction times. (C) Western blot analysis of DRG explants treated with DMSO (control), CH02 (20 μM), CH02+BGJ398, or BGJ398 (2 μM) for 4 h to examine phospho-FGFR levels after 6 days in ex vivo culture. Tubulin antibodies were used as the loading control. (D-F) Quantification of p-FGFR expression levels in panels A, B, and C. n = 3 independent experiments. Relative protein expression levels were quantified after normalization to FGFR2 or FGFR1. Values are means ± SEM. (G) Representative images of the CH02 effect on axonal growth in ex vivo DRG explants cultured for 1 day and treated with DMSO (control), CH02 (20 μM), CH02+BGJ398, or BGJ398 (2 μM) for 4 days. DRG explants were fixed in 4% paraformaldehyde and immunostained for NF200 antibodies. Scale bar = 100 μm. (H) Quantification of axon growth in DRG explants (G) using Neurite-J software (n = 5 for each condition; *control vs. CH02; #control vs. BGJ398; ** p < 0.01, *** p < 0.001, **** p < 0.0001, # p < 0.05, ## p < 0.01 by two-way ANOVA with Dunnett's test; mean ± SEM). (I) Representative images of the CH02 effect on axonal growth in primary cultured DRG neurons treated with DMSO (control), CH02 (20 μM), CH02+BGJ398, orBGJ398 (2 μM) for 48 h after 12- h of culture. Subsequently, DRG neurons were fixed in 4% paraformaldehyde and immunostained with β-tubulin antibodies. Images of random neurons were acquired by fluorescence microscopy. Scale bar = 25 μm. (J) Quantification of the longest axon length of each neuron randomly selected from (I) using Image-Pro Plus 6.0 software (n = 100 for each condition; * p < 0.05, ** p < 0.01, **** p < 0.0001 by one-way ANOVA with Dunnett's; ns, not significant; mean ± SD).

Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti-phospho-FGFR (1:1000; CST); anti-FGFR2 (1:1000; Abcam), rabbit monoclonal anti-FGFR1(1:1000; CST), rabbit monoclonal anti-phospho-VEGFR2 (1:1000; CST), rabbit monoclonal anti-VEGFR2 (1:1000; CST), rabbit polyclonal anti-phospho-ERBB2 (1:1000; Abcam), rabbit monoclonal anti-ERBB2 (1:1000; Abcam), rabbit monoclonal anti-phospho-MET (1:1000; CST), rabbit monoclonal anti-MET (1:1000; Abcam), rabbit monoclonal anti-phospho- PDGFR beta (1:1000; Abcam), rabbit monoclonal anti-PDGFR beta (1:1000; Abcam), rabbit monoclonal anti-phospho-ERK1/2 (1:1000; CST), rabbit monoclonal anti-ERK1/2 (1:1000; CST), rabbit monoclonal anti-phospho-AKT (1:1000; CST), rabbit monoclonal anti-AKT (1:1000; CST), rabbit monoclonal anti-mTOR (1:1000; CST), rabbit monoclonal anti-phospho-mTOR (1:1000; CST), rabbit monoclonal anti-β-Tubulin (1:1000; CST), and rabbit monoclonal anti- GAPDH (1:1000; CST).

Techniques: Western Blot, Expressing, Ex Vivo, Cell Culture, Software, Fluorescence, Microscopy