Journal: PLoS ONE

Article Title: Targeting Wild-Type and Mutationally Activated FGFR4 in Rhabdomyosarcoma with the Inhibitor Ponatinib (AP24534)

doi: 10.1371/journal.pone.0076551

Figure Lengend Snippet: (A) The sensitivity of a panel of six fusion-positive RMS cell lines (RH4, RH28, JR, RH41, RH5, and RH30) and eight fusion-negative RMS cell lines (BIRCH, RH18, TTC-442, CT-10, CTR, TTC-516, RD, and RH36) to ponatinib is correlated to FGFR4 mRNA expression levels by Spearman ranking (p = 0.0261). (B) Comparing the variation in IC 50 values of fusion-positive (FP) and fusion-negative (FN) RMS cell lines shows a significant difference by F test (p = 0.0125). (C) A difference in IC 50 values can be seen between RMS cell lines expressing low (below a relative level of 6) and high (above a relative level of 6) levels of FGFR4 (p = 0.0344).

Article Snippet: For FGFR4 autophosphorylation immunoblots, 200–500 μg of protein lysate, as determined by BCA protein assay (Pierce), was first immunoprecipitated with a FGFR4 antibody (sc-124; Santa Cruz Biotechnology) and then incubated overnight with protein A/G agarose beads.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Targeting Wild-Type and Mutationally Activated FGFR4 in Rhabdomyosarcoma with the Inhibitor Ponatinib (AP24534)

doi: 10.1371/journal.pone.0076551

Figure Lengend Snippet: RMS772 cell harboring activating FGFR4 mutations V550E or N535K are more sensitive to ponatinib (AP24534) after 24 hour treatment than RMS772 cells expressing wild-type (WT) FGFR4 or the empty vector (VCtrl) (*p = <0.0001, **p = <0.0001).

Article Snippet: For FGFR4 autophosphorylation immunoblots, 200–500 μg of protein lysate, as determined by BCA protein assay (Pierce), was first immunoprecipitated with a FGFR4 antibody (sc-124; Santa Cruz Biotechnology) and then incubated overnight with protein A/G agarose beads.

Techniques: Expressing, Plasmid Preparation

Journal: PLoS ONE

Article Title: Targeting Wild-Type and Mutationally Activated FGFR4 in Rhabdomyosarcoma with the Inhibitor Ponatinib (AP24534)

doi: 10.1371/journal.pone.0076551

Figure Lengend Snippet: (A) Cell cycle analysis of the two most sensitive cell lines to ponatinib, RH4 and RH5, and the two RMS772 cell lines expressing FGFR4 mutations (N535K and V550E) showed increased time in sub G 1 phase and decreased time in S phase across all four cell lines after 24 hours of treatment with 2.5 µM ponatinib. (B) Cell death induced by 2.5 µM ponatinib treatment is mediated via the caspase 3/7 pathway (*p = 0.0029, **p = 0.0027, ***p = 0.0017, ****p = 0.0001).

Article Snippet: For FGFR4 autophosphorylation immunoblots, 200–500 μg of protein lysate, as determined by BCA protein assay (Pierce), was first immunoprecipitated with a FGFR4 antibody (sc-124; Santa Cruz Biotechnology) and then incubated overnight with protein A/G agarose beads.

Techniques: Cell Cycle Assay, Expressing

Journal: PLoS ONE

Article Title: Targeting Wild-Type and Mutationally Activated FGFR4 in Rhabdomyosarcoma with the Inhibitor Ponatinib (AP24534)

doi: 10.1371/journal.pone.0076551

Figure Lengend Snippet: (A) A dose-dependent decrease in wild-type FGFR4 phosphorylation as shown by immunoprecipitation of FGFR4 and immunoblotting for phosphotyrosine. (B) A similar dose-dependent inhibition is seen for FGFR4 with the V550E and N535K mutation. (C-D) Western blot shows a dose-dependent decrease in STAT3 phosphorylation after treatment with ponatinib for three fusion-positive (RH4, RH5, and RH41) and one fusion-negative (CTR) RMS cell lines as well as the two RMS772 cell lines expressing the FGFR4 mutations N535K and V550E.

Article Snippet: For FGFR4 autophosphorylation immunoblots, 200–500 μg of protein lysate, as determined by BCA protein assay (Pierce), was first immunoprecipitated with a FGFR4 antibody (sc-124; Santa Cruz Biotechnology) and then incubated overnight with protein A/G agarose beads.

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Inhibition, Mutagenesis, Expressing

Journal: PLoS ONE

Article Title: Targeting Wild-Type and Mutationally Activated FGFR4 in Rhabdomyosarcoma with the Inhibitor Ponatinib (AP24534)

doi: 10.1371/journal.pone.0076551

Figure Lengend Snippet: Arrow indicates the start of treatment. (A) Treatment of tumors harboring the FGFR4 N535K mutation with ponatinib significantly inhibits tumor growth after 10 days of treatment (*p = 0.0165, **p = 0.0048). (B) Treatment of tumors containing the FGFR4 V550E mutation with ponatinib significantly inhibits tumor growth after 6 days of treatment (*p = 0.0185, **p = 0.0087, ***p = 0.0005). (C) Treatment of tumors expressing the wild-type FGFR4 with ponatinib does not affect tumor growth. (D) Treatment of tumors expressing the empty vector with ponatinib does not affect tumor growth.

Article Snippet: For FGFR4 autophosphorylation immunoblots, 200–500 μg of protein lysate, as determined by BCA protein assay (Pierce), was first immunoprecipitated with a FGFR4 antibody (sc-124; Santa Cruz Biotechnology) and then incubated overnight with protein A/G agarose beads.

Techniques: Mutagenesis, Expressing, Plasmid Preparation

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