Journal: Theranostics

Article Title: Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis

doi: 10.7150/thno.72269

Figure Lengend Snippet: High pY levels might affect the proliferation, migration and differentiation of lung fibroblasts. A , Changes in cell proliferation between hnLFs and hfLFs determined by CCK-8 kit (n = 7). *** P < 0.001. B-C , Assessing cell proliferation by EdU assay between hnLFs and hfLFs (n = 6). *** P < 0.001. D-G , Comparison of migration between hnLFs and hfLFs (n = 6). *** P < 0.001. H , Representative images of hnLFs and hfLFs under TEM. Arrows pointed to the lipid droplets in cytoplasm. I , Representative fluorescence images of Nile Red in hnLFs and hfLFs. J-K , The contractility of hnLFs and hfLFs in 3D collagen matrices (n = 6). *** P < 0.001. L , Phospho-RTK array analysis with 200 μg of lysates from hnLFs and hfLFs, which were serum-starved for 6 h. M , Quantification of phospho-RTK levels (average of two drops fold average of positive control). N-O , Western blot and quantification of p-VEGR2, p-PDGFRβ, p-EGFR and p-FGFR1 in hnLFs and hfLFs (n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001. P-Q , Western blot and quantification of p-PLCγ1, p-GAB1, p-SHC and p-SRC in hnLFs and hfLFs (n = 6). ** P < 0.01, *** P < 0.001. R-S , Western blot and quantification of p-AKT, p-ERK1/2 and p-STAT3 in hnLFs and hfLFs (n = 6). ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against FN (Cat#15613-1-AP), COL1A1 (Cat#67288-1-lg), α-SMA (Cat#55135-1-AP, Cat#67735-1-AP), FGFR1 (Cat#60325-1-lg), CD31 (Cat#60287-1-lg), CD45 (Cat#11265-1-lg), EpCAM (Cat#60287-1-lg), Vimentin (Cat#10366-1-AP) and GAPDH (Cat#60004-1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Migration, CCK-8 Assay, EdU Assay, Comparison, Fluorescence, Positive Control, Western Blot

Journal: Theranostics

Article Title: Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis

doi: 10.7150/thno.72269

Figure Lengend Snippet: SH2 superbinder blocked multiple fibrosis associated pathways through interrupting pY-SH2 combination in pY mediated signal transmission. A-B , Phospho-RTK array analysis with 200 μg of lysates from hfLFs treated with 1 μM GST-SH2 WT or GST-SH2 TrM for 24 h. C , Western blot of p-VEGR2, p-PDGFRβ, p-EGFR and p-FGFR1 in hnLFs and hfLFs after GST, GST-SH2 WT and GST-SH2 TrM incubation. D , Western blot of p-PLCγ1, p-GAB1, p-SHC and p-SRC in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM incubation. E , Western blot of p-AKT, p-ERK1/2 and p-STAT3 in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM incubation. F , hnLFs and hfLFs were incubated with 1 μM GST, GST-SH2 WT or GST-SH2 TrM for 24 h. The immunoprecipitation of EGFR and SHC was detected. G , Representative fluorescence images of EGFR and SHC colocalization in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM treatment. H , Immunoprecipitations of GRB2 with GAB1, SHC, EGFR and PDGFRβ. I , GST pull down assay showed the binding capacity of SH2 superbinder with pY in hnLFs and hfLFs. J , Representative fluorescence images of GST tag and pY colocalization in hnLFs and hfLFs after incubation with GST, GST-SH2 WT or GST-SH2 TrM. K-L , GST pull down assay showed the binding capacity of SH2 superbinder with RTKs (such as VEGFR2, PDGFRβ, EGFR and FGFR1) and adaptor proteins (such as GAB1, SHC and SRC) in hfLFs and hfLFs.

Article Snippet: Antibodies against FN (Cat#15613-1-AP), COL1A1 (Cat#67288-1-lg), α-SMA (Cat#55135-1-AP, Cat#67735-1-AP), FGFR1 (Cat#60325-1-lg), CD31 (Cat#60287-1-lg), CD45 (Cat#11265-1-lg), EpCAM (Cat#60287-1-lg), Vimentin (Cat#10366-1-AP) and GAPDH (Cat#60004-1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Transmission Assay, Western Blot, Incubation, Immunoprecipitation, Fluorescence, Pull Down Assay, Binding Assay

Journal: Journal of Cancer

Article Title: mRNA Expression of FGFR1 as Potential Marker for Predicting Prognosis of Surgical Resection of Small Cell Lung Cancer may be better than Protein Expression and Gene Amplification.

doi: 10.7150/jca.44476

Figure Lengend Snippet: Figure 1. Diagrammatic representation of FGFR1 mutation from www.cbioportal.org.

Article Snippet: IHC for FGFR1 was performed using FGFR1 antibody (Cat, #BA0485, Boster Biological Technology Co. Ltd).

Techniques: Mutagenesis

Journal: Journal of Cancer

Article Title: mRNA Expression of FGFR1 as Potential Marker for Predicting Prognosis of Surgical Resection of Small Cell Lung Cancer may be better than Protein Expression and Gene Amplification.

doi: 10.7150/jca.44476

Figure Lengend Snippet: Figure 2. FGFR1 protein expression by immunohistochemistry in SCLC and their correlations with prognosis. (A) shows FGFR1 protein positive expression; (B) shows FGFR1 protein negative expression. Kaplan–Meier Survival analysis of FGFR1 protein-positive vs. FGFR1 protein-negative (C and D).

Article Snippet: IHC for FGFR1 was performed using FGFR1 antibody (Cat, #BA0485, Boster Biological Technology Co. Ltd).

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Cancer

Article Title: mRNA Expression of FGFR1 as Potential Marker for Predicting Prognosis of Surgical Resection of Small Cell Lung Cancer may be better than Protein Expression and Gene Amplification.

doi: 10.7150/jca.44476

Figure Lengend Snippet: Figure 3. FGFR1 amplification by fluorescence in situ hybridization in SCLC and their correlations with prognosis. (A) shows FGFR1 amplification (B) shows FGFR1 non-amplification. Kaplan–Meier Survival analysis of FGFR1 amplified vs. non-amplified tumors (C and D).

Article Snippet: IHC for FGFR1 was performed using FGFR1 antibody (Cat, #BA0485, Boster Biological Technology Co. Ltd).

Techniques: Amplification, Fluorescence, In Situ Hybridization

Journal: Oncotarget

Article Title: FGFR1β is a driver isoform of FGFR1 alternative splicing in breast cancer cells

doi: 10.18632/oncotarget.26530

Figure Lengend Snippet: (A-D) Bioinformatic analysis of expression of FGFR1 splicing variants in TCGA breast cancer samples. (A) Expression of FGFR1 variants in FGFR1-amplified and non-amplified samples. # : p=3.69e-19 (amp vs non-amp); * : p=7.35e-15 (amp vs non-amp); #* : p=0.721 (amp vs non-amp). (B) FGFR1 amplification frequency in subtypes. FGFR1 copy numbers were analyzed for amplification frequency in 3 groups of samples – basal, HER2+ and luminal subtypes. (C) Expression of FGFR1α in 3 subtype groups. * : p=0.0305 (basal vs luminal). HER2 + vs luminal: p=0.105; basal vs HER2 + : p=0.669. (D) Expression of FGFR1β in 3 subtype groups. * : p=0.0016 (basal vs luminal). HER2 + vs luminal: p=0.812; basal vs HER2 + : p=0.0725. (E) Immunoblotting of FGFR1 in the cell lines. Cell lysates were prepared from 15 breast cancer cell lines and analyzed by SDS-PAGE. FGFR1α and FGFR1β proteins were detected by anti-FGFR1 antibody. The first lane on the left was the same lane of MDA-MB-134VI cells with lighter exposure. (F) Subtypes of breast cancer cell lines. (G) RelativeFGFR1β levels in cell lines. The relative FGFR1β levels were obtained by normalizing with β-actin. * : p=0.0065 (basal vs luminal); #: p=0.0003 (HER2 + vs luminal). (H) FGFR1β/FGFR1α ratio in cell lines. FGFR1β and FGFR1α expression levels in WB were quantitated by ImageJ software. The FGFR1-β/FGFR1α ratio was present in each subtype groups. * : p<0.01 (basal vs luminal); #: p<0.001 (HER2 + vs luminal).

Article Snippet: To create FGFR1α and FGFR1β variants, MCF-10A cells were infected with virus packaged from 293 cells using viral expression vectors pLenti-C-Myc-FGFR1α (#RC202080L1) and pLenti-C-Myc-FGFR1β (#RC210629L1) plasmids as well as empty vector (#PS100064) (OriGene, Rockville, MD).

Techniques: Expressing, Amplification, Western Blot, SDS Page, Software

Journal: Oncotarget

Article Title: FGFR1β is a driver isoform of FGFR1 alternative splicing in breast cancer cells

doi: 10.18632/oncotarget.26530

Figure Lengend Snippet: (A) Viral FGFR1 expression. Virus particles were packaged for FGFR1α and FGFR1β, and were infected into MCF-10A cells. Expression of FGFR1 was confirmed by immunoblotting with anti-FGFR1 antibody. (B, C) Immunoblotting of FGFR signaling activity. MCF-10Acells were treated with FGF2 at 20ng/ml or vehicle control for 24 hours. The MAPK pathway was detected in the cell lysates with antibodies against phospho-MEK1/2, phospho-ERK1/2 (B), while the PI3K pathway was detected with antibodies against phospho-pAKT, phospho-S6 and phospho-4E-BP1 (C). (D) Cell growth rate. MCF-10A cells expressing FGFR1α, FGFR1β, and vector were cultured in 96-well plates in a normal condition for the indicated days. Cell viability was measured by SRB staining. Cell proliferation rate was calculated by normalizing OD490nm values to day 1 OD value. # : p<0.05 vs empty vector; * : p<0.01 vs empty vector. (E-G) Cell colony formation. MCF-10A cells seeded in 6-well plates were cultured for 3 weeks followed by crystal violet staining (E). Total colony area (F) and average colony size (G) were quantitated using ImageJ software. Average values were calculated from triplicate wells for each group. * : p<0.01 vs empty vector.

Article Snippet: To create FGFR1α and FGFR1β variants, MCF-10A cells were infected with virus packaged from 293 cells using viral expression vectors pLenti-C-Myc-FGFR1α (#RC202080L1) and pLenti-C-Myc-FGFR1β (#RC210629L1) plasmids as well as empty vector (#PS100064) (OriGene, Rockville, MD).

Techniques: Expressing, Infection, Western Blot, Activity Assay, Plasmid Preparation, Cell Culture, Staining, Software

Journal: Oncotarget

Article Title: FGFR1β is a driver isoform of FGFR1 alternative splicing in breast cancer cells

doi: 10.18632/oncotarget.26530

Figure Lengend Snippet: (A) Three-dimensional Matrigel assay. MCF-10A cells expressing FGFR1α, FGFR1β, and vector were seeded into chamber wells coated with Matrigel in MEGM medium containing FGF2 at 20ng/ml and BGJ-398 at 2μM for 3 weeks. Phase-contrast micrographs of spherical mammary structure were captured. (B) Soft agar assay. MCF-10A cells were seeded into 3.5% agar gel and cultured for 3 weeks. Anchorage-independent colonies were visualized by Iodonitrotetrazolium chloride staining. (C) E-cadherin immunoblotting. MCF-10A cells were treated with TGF-β1 at 5ng/ml or vehicle control for 2 days. Cell lysates were analyzed by western blot using anti-E-cadherin antibody with a normalization by β-actin.

Article Snippet: To create FGFR1α and FGFR1β variants, MCF-10A cells were infected with virus packaged from 293 cells using viral expression vectors pLenti-C-Myc-FGFR1α (#RC202080L1) and pLenti-C-Myc-FGFR1β (#RC210629L1) plasmids as well as empty vector (#PS100064) (OriGene, Rockville, MD).

Techniques: Matrigel Assay, Expressing, Plasmid Preparation, Soft Agar Assay, Cell Culture, Staining, Western Blot

Journal: Oncotarget

Article Title: FGFR1β is a driver isoform of FGFR1 alternative splicing in breast cancer cells

doi: 10.18632/oncotarget.26530

Figure Lengend Snippet: (A) Cell survival screening. Cell lines seeded in 96-well plates were incubated with BGJ-398 at a serial dilutions for 3 days, followed cell viability measurement by SRB. IC50s of cell survival inhibition were calculated using GraphPad Prism7 software. (B) FGFR1β levels in cell lines measured by WB in Figure . (C) FGFR1β/FGFR1α expression ratio in the cell lines measured by WB in Figure . (D) BGJ398 IC50s in cell line groups with high and low FGFR1β levels and FGFR1β/FGFR1α ratio with thresholds 0.05 and 0.9 respectively. * : p=0.015; #: p=0.0033 (low vs high). (E, F) Correlations between BGJ-398 IC50 and absolute FGFR1β levels (E) or FGFR1β/FGFR1α ratio (F). The correlation analysis for Pearson r value was performed using GraphPad Prizm7 software. (G) MFM-223 cells were treated with Debio-1347 or BGJ-398 at 2uM for 3 days. Expression of FGFR1 and PTBP1 were detected by immunoblotting with anti-FGFR1 and anti-PTBP1 antibodies. (H) PTBP1 knockdown. MFM-223 cells were infected with PTBP1 shRNA virus or control shRNA vector. FGFR1 and PTBP1 were detected by immunoblotting. (I) PTBP1-deficient cells and control shRNA cells were incubated with BGJ-398 at 2uM or vehicle controls for 3 days, followed by immunoblotting. (J) Colony formation of PTBP1-deficient cells. The control and PTBP1-knockdown MFM-223 cells were cultured for 3 weeks for colony formation. Total colony area was quantitated by ImageJ. * : p=0.0136.

Article Snippet: To create FGFR1α and FGFR1β variants, MCF-10A cells were infected with virus packaged from 293 cells using viral expression vectors pLenti-C-Myc-FGFR1α (#RC202080L1) and pLenti-C-Myc-FGFR1β (#RC210629L1) plasmids as well as empty vector (#PS100064) (OriGene, Rockville, MD).

Techniques: Incubation, Inhibition, Software, Expressing, Western Blot, Infection, shRNA, Plasmid Preparation, Cell Culture

Journal: Oncotarget

Article Title: FGFR1β is a driver isoform of FGFR1 alternative splicing in breast cancer cells

doi: 10.18632/oncotarget.26530

Figure Lengend Snippet: (A) RT-PCR of FGFR1α and FGFR1β. MDA-MB-134VI cells were cultured for 3 days with hormone-deprived FBS, then treated with 17β-estradiol at 0.1μM (E), 4-hydroxytamoxifen at 1μM (T), or both (E+T), or vehicle (V) for 2 days. RT-PCR was performed to detect mRNAs of FGFR1α, FGFR1β, and GAPDH. (B) Immunoblotting. The MDA-MB-134VI cells cultured with hormone-deprived FBS or normal FBS for 3 days were incubated with 17β-estradiol or 4-hydroxytamoxifen treatment respectively for 2 days. E1 and E2: 17β-estradiol at 0.1 and 0.5μM; T1 and T2: 4-hydroxytamoxifen at 1 and 5μM; V: vehicle. (C) Ratio of quantitated FGFR1β/FGFR1α expression in Figure . (D) WB of FGFR1 and PTBP1. MDA-MB-134VI cells were treated with 17β-estradiol (E2) at 0.1μM or vehicle control for 2 days, or infected with PTBP1 shRNA virus or control shRNA. Expression of FGFR1 and PTBP1 were detected with anti-FGFR1 and anti-PTBP1 antibodies. (E) Effects of drug combination on cell survival. MDA-MB-134VI cells seeded in 96-well plates were incubated with single or combination of 4-hydroxytamoxifen and BGJ-398 at doses from 0.05-20000nM and 1250-20000nM respectively for 5 days. Cell survival rate was measured by SRB assay. IC50 and combination index (CI) were calculated using CompuSyn software. (CI<1: synergy; CI>1: antagonism) (F) Colony formation assay. MDA-MB-134VI cells were cultured in the presence of BGJ-398 and 4-OHT at 1nM and 0.1nM respectively and their combination for 4 weeks. Colony formation was visualized by crystal violet staining. Total colony area was quantitated using ImageJ.

Article Snippet: To create FGFR1α and FGFR1β variants, MCF-10A cells were infected with virus packaged from 293 cells using viral expression vectors pLenti-C-Myc-FGFR1α (#RC202080L1) and pLenti-C-Myc-FGFR1β (#RC210629L1) plasmids as well as empty vector (#PS100064) (OriGene, Rockville, MD).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Western Blot, Incubation, Expressing, Infection, shRNA, Sulforhodamine B Assay, Software, Colony Assay, Staining