Journal: Cancers

Article Title: The Receptor Tyrosine Kinase RON and Its Isoforms as Therapeutic Targets in Ewing Sarcoma

doi: 10.3390/cancers12040904

Figure Lengend Snippet: RON is expressed in Ewing sarcomas and cell lines. ( a ) Relative RON transcript expression in Ewing sarcoma primary tumors from patients with localized (non-met) or metastatic (met) disease in comparison to MSC cultures, as determined by qPCR. ( b ) Respective RON expression in Ewing sarcoma cell lines (EwS) compared to MSC cultures. ( c ) RON protein is expressed and phosphorylated in Ewing sarcoma and rhabdomyosarcoma (RMS) cell lines. Cells were grown in standard tissue culture conditions. Following analysis of phospho-RON, blots were stripped and re-probed for total RON expression; 10% gel; numbers indicate densitometry readings relative to respective actin loading control.

Article Snippet: Primary antibodies detecting RON were Cat-No. HPA007657 (SEMA domain amino acids 283-433, corresponding to exons 1-2; unless otherwise specified, this antibody was used for total RON detection) and Cat-No. HPA008180 (IPT3 domain amino acids 767–875, corresponding to exons 9–10) from Sigma-Aldrich; phospho-RON (kinase domain Tyr1238/39) (Cat-No. AF1947) was from R&D Systems; MET (25H2) (Cat-No. 3127), phospho-MET (Tyr1234/35; D26) (Cat-No. 3077) and phospho-IGF1R (Tyr1131/1146) (Cat-No. 3021) were from Cell Signaling Technology (Beverly, MA, USA); IGF1Rβ (C20) (Cat-No. sc-713) and actin (C4) (Cat-No. sc-47778) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Comparison, Control

Journal: Cancers

Article Title: The Receptor Tyrosine Kinase RON and Its Isoforms as Therapeutic Targets in Ewing Sarcoma

doi: 10.3390/cancers12040904

Figure Lengend Snippet: Ewing sarcomas express targeting-relevant RON isoforms. ( a ) Western blots suggest the presence of full-length RON (flRON) splice variants in pediatric sarcoma cell lines. RON protein expression was analyzed in comparison to characterized isoforms in HT-29 and HCT-116. Cells were grown in standard tissue culture conditions. Following analysis of phospho-RON, Western blots were stripped and re-probed for analysis of two distinct total RON antibodies directed at the SEMA and IPT3 domain epitopes. Arrows indicate RON species. 8% gel. ( b ) Ewing sarcomas express the short-form RON ( sfRON ) isoform containing (upper band) and/or lacking intron 11 sequences (lower band). Tumor samples are numbered; M indicates primary tumors from patients with metastatic disease, R indicates a relapsed tumor. ( c ) Sarcoma cell lines express sfRON . RT-PCR was performed on mRNA isolated from cell lines grown in standard conditions. ( d , e ) Treatment with 5-Aza-2’-deoxycytidine (5-Aza-CdR) modulates flRON ( d ) and sfRON ( e ) transcription. RT-PCRs were performed on mRNA isolated from cell lines grown in standard conditions and treated with 2.5 µM 5-Aza-CdR for 72 h where indicated. In ( a – e ), numbers indicate densitometry readings relative to the respective actin or GAPDH loading control.

Article Snippet: Primary antibodies detecting RON were Cat-No. HPA007657 (SEMA domain amino acids 283-433, corresponding to exons 1-2; unless otherwise specified, this antibody was used for total RON detection) and Cat-No. HPA008180 (IPT3 domain amino acids 767–875, corresponding to exons 9–10) from Sigma-Aldrich; phospho-RON (kinase domain Tyr1238/39) (Cat-No. AF1947) was from R&D Systems; MET (25H2) (Cat-No. 3127), phospho-MET (Tyr1234/35; D26) (Cat-No. 3077) and phospho-IGF1R (Tyr1131/1146) (Cat-No. 3021) were from Cell Signaling Technology (Beverly, MA, USA); IGF1Rβ (C20) (Cat-No. sc-713) and actin (C4) (Cat-No. sc-47778) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Expressing, Comparison, Reverse Transcription Polymerase Chain Reaction, Isolation, Control

Journal: Genes

Article Title: Single-Cell Transcriptomic Analysis of the Mouse Pancreas: Characteristic Features of Pancreatic Ductal Cells in Chronic Pancreatitis

doi: 10.3390/genes13061015

Figure Lengend Snippet: Subpopulation identification of pancreatic mesenchymal cells in normal mice. ( A ) t-SNE illustration of four subgroups identified from sub-clustering of the mesenchymal cell population, colored according to the cluster assignments (left panel). Tables demonstrating the top differentially expressed genes when comparing one subcluster to all other mesenchymal cells (right panel). ( B ) Violin plots showing representative marker genes in the subgroups. ( C ) Representative GO terms enriched for marker genes in Mes.4 based on a functional enrichment analysis. ( D , E ) Representative immunohistochemical images of pancreatic tissues from the control mice stained for Fn1 (red arrow) and Cxcl14 (black arrow). Scale bar = 50 μm.

Article Snippet: Immunohistochemical staining was applied to the paraffin sections with antibodies against CD45 (ab10558, Abcam), F4/80 (#70076, Cell Signaling Technology, Danvers, MA, USA), CD3 (ab16669, Abcam), CD20 (MA5-13141, Invitrogen, Carlsbad, CA, USA), Cxcl14 (MAB866, Novus, Littleton, CO, USA), FN1 (15613-1-AP, Proteintech, Wuhan, China), MMP7 (10374-2-AP, Proteintech), and TTR (11891-1-AP, Proteintech).

Techniques: Marker, Functional Assay, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: MST1R/RON and EGFR in a complex with syndecans sustain carcinoma S-phase progression by preventing p38MAPK activation

doi: 10.1101/252742

Figure Lengend Snippet: A. UM-SCC47 cells were treated for 72 hr with either control siRNA, or siRNA specific for human Sdc4, β4 integrin (ITGB4), EGFR, or α3 integrin (ITGA3), followed by addition of EdU for 45 min to monitor DNA synthesis prior to fixation and staining; Western blots showing individual receptor expression 72 hr after siRNA transfection are shown (inset); B . Either NOKs or UM-SCC47 cells were allowed to invade through LN332-coated filters for 16 hr in serum-free conditions following stimulation with 10 ng/ml EGF +/− 30 μM SSTN EGFR or vehicle; C. Quantification of NOK or UM-SCC47 cell invasion as in ( B ) in the presence or absence of the EGFR kinase inhibitors gefitinib (3 μM) or erlotinib (2 μM), SFK inhibitor PP2 or its inactive analogue PP3 (3 μM), MST1R/RON inhibitors CAS 913376-84-8 (1 μM) or BMS-0777607 (3 μM), c-Abl inhibitor GNF5 (2 μM), pan-p38MAPK inhibitor BIRB-796 (100 nM) and α6β4 (3E1) or α3β1 (P1B5) integrin blocking antibody (10 μg/ml); D. DNA synthesis is detected by EdU incorporation in UM-SCC47 cells grown for 3 hr in medium containing SSTN EGFR , the EGFR kinase inhibitors gefitinib or erlotinib, or SFK inhibitor PP2 versus its inactive analogue PP3.

Article Snippet: Western blotting primary antibodies include: goat polyclonal human Sdc4 (AF2918, 0.5 μg/mL), EGFR (AF231, 1 μg/mL) and total MST1R/RON kinase (AF691, 1 μg/mL); rabbit polyclonal pY1238/1239 MST1R/RON (AF1947, 1 μg/mL) and mouse human ITGB4 mAb 422325 (1 μg/mL) from R&D Systems (Minneapolis, MN); rabbit polyclonal ITGA3 (1:250) from Novus Biologicals (Littleton, CO); rabbit mAbs 73E5 (c-Abl pY245, 1 μg/mL), 247C7 (c-Abl pY412, 1 μg/mL), D13E1 (total p38MAPK, 1:1000), 11H10 (tubulin, 1:1000), 133D3 (pS345-CHK1, 1:1000), rabbit polyclonal pT68-CHK2 (2661S, 1:1000) and pT183/Y185 p38MAPK mouse mAb 28B10 (1:2000) from Cell Signaling Technology (Danvers, MA); mouse mAbs 8E9 (total c-Abl, 1 μg/mL) and TU-01 (tubulin, 1:1000), and rabbit polyclonal Sdc2 (1 μg/mL) from Invitrogen/ThermoFisher Scientific (Rockford, IL); p53 mouse mAb DO-1 (1:750) from Santa Cruz Biotechnology (Dallas, TX) and mouse mAbs from Millipore-Sigma, JBW301 (pS139-γH2AX, 1:1000) and AC-74 (β-actin, 1:5000).

Techniques: DNA Synthesis, Staining, Western Blot, Expressing, Transfection, Blocking Assay

Journal: bioRxiv

Article Title: MST1R/RON and EGFR in a complex with syndecans sustain carcinoma S-phase progression by preventing p38MAPK activation

doi: 10.1101/252742

Figure Lengend Snippet: A, B. Select neoplastic, preneoplastic and non-transformed epithelial cells were treated for 3 hr in DMSO (vehicle), the MST1R/RON inhibitors CAS 913376-84-8 (1 μM) or BMS-777607 (3 μM) or the Abl kinase inhibitors GNF5 (2 μM) or PPY-A (0.2 μM). EdU was added for the last 45 min. The cells were fixed and extracted to stain for EdU incorporation ( A ) or DNA-bound PCNA ( B ); C. UM-SCC47 cells treated with vehicle alone or 30 μM SSTN EGFR for 3 hr were lysed and subjected to immunoprecipitation with non-specific isotype control IgG or mAb 8G3 to Sdc4. Immunoprecipitates were probed for the presence of EGFR, α3 integrin (ITGA3), β4 integrin (ITGB4), total and active MST1R/RON (pY1238/1239), total and active c-Abl (pY412 and pY245), Sdc2 and Sdc4; D . UM-SCC47 cells were treated for 3 hr with vehicle or 30 μM SSTN EGFR , then extracted and analyzed on western blots for active pY245 or pY412 phosphorylated c-Abl.

Article Snippet: Western blotting primary antibodies include: goat polyclonal human Sdc4 (AF2918, 0.5 μg/mL), EGFR (AF231, 1 μg/mL) and total MST1R/RON kinase (AF691, 1 μg/mL); rabbit polyclonal pY1238/1239 MST1R/RON (AF1947, 1 μg/mL) and mouse human ITGB4 mAb 422325 (1 μg/mL) from R&D Systems (Minneapolis, MN); rabbit polyclonal ITGA3 (1:250) from Novus Biologicals (Littleton, CO); rabbit mAbs 73E5 (c-Abl pY245, 1 μg/mL), 247C7 (c-Abl pY412, 1 μg/mL), D13E1 (total p38MAPK, 1:1000), 11H10 (tubulin, 1:1000), 133D3 (pS345-CHK1, 1:1000), rabbit polyclonal pT68-CHK2 (2661S, 1:1000) and pT183/Y185 p38MAPK mouse mAb 28B10 (1:2000) from Cell Signaling Technology (Danvers, MA); mouse mAbs 8E9 (total c-Abl, 1 μg/mL) and TU-01 (tubulin, 1:1000), and rabbit polyclonal Sdc2 (1 μg/mL) from Invitrogen/ThermoFisher Scientific (Rockford, IL); p53 mouse mAb DO-1 (1:750) from Santa Cruz Biotechnology (Dallas, TX) and mouse mAbs from Millipore-Sigma, JBW301 (pS139-γH2AX, 1:1000) and AC-74 (β-actin, 1:5000).

Techniques: Transformation Assay, Staining, Immunoprecipitation, Western Blot

Journal: Molecular Cancer

Article Title: Loss of FBXW7-mediated degradation of BRAF elicits resistance to BET inhibitors in adult T cell leukemia cells

doi: 10.1186/s12943-020-01254-x

Figure Lengend Snippet: Phenotypic characterization of FBXW7 mutants

Article Snippet: Endogenous expression of proteins was performed using antibodies for CyclinE (Santa-Cruz #sc-198), Mcl-1 (Santa-Cruz #sc-12,756), c-myc (9E10), FBXW7 (Proteintech #28424–1-AP), BRAF (Santa-cruz #sc-5284), p-MEK-1/2 (Calbiochem), p-ERK-1/2, and STAT3 (Santa-Cruz #sc-482).

Techniques:

Journal: Molecular Cancer

Article Title: Loss of FBXW7-mediated degradation of BRAF elicits resistance to BET inhibitors in adult T cell leukemia cells

doi: 10.1186/s12943-020-01254-x

Figure Lengend Snippet: Resistance to BET inhibitors is linked to activation of RAF/MEK/ERK signaling pathway by FBXW7 mutants. a RPPA was used to comprehensively analyze the protein expression in MT1 cells expressing FBXW7 WT and S462P. BRAF is up-regulated in MT1 FBXW7 S462P expressing cells compared to MT1 cells expressing FBXW7 WT. b Cell signaling pathways affected by wild-type or mutant FBXW7 S462P. ( c ) Gene ontology fold enrichment from MT1 cells expressing FBXW7 WT, S462P, R465C, R505C and R479Q. d MT1 cells stably expressing doxycycline inducible FBXW7 WT or S462P mutant were treated with DMSO or JQ1 in the presence or absence of Dox and analyzed for expression of BRAF, p-MEK, p-ERK, c-MYC and STAT3 by Western blot using actin as a loading control. e The expression of Myc and BRAF in HTLV-I/ATL-lines was analyzed by WB. Correlation between Myc/BRAF expression and IC50 was calculated by Pearson correlation calculations. JQ1 IC50 in relation to c-MYC or BRAF expression was calculated in ATL-lines treated with different concentration of JQ1 or dimethyl sulfoxide (DMSO) for 72 h. Each cell line was treated at least twice for standard deviation. Cell proliferation was analyzed by XTT assay (Roche)

Article Snippet: Endogenous expression of proteins was performed using antibodies for CyclinE (Santa-Cruz #sc-198), Mcl-1 (Santa-Cruz #sc-12,756), c-myc (9E10), FBXW7 (Proteintech #28424–1-AP), BRAF (Santa-cruz #sc-5284), p-MEK-1/2 (Calbiochem), p-ERK-1/2, and STAT3 (Santa-Cruz #sc-482).

Techniques: Activation Assay, Expressing, Mutagenesis, Stable Transfection, Western Blot, Concentration Assay, Standard Deviation, XTT Assay

Journal: Molecular Cancer

Article Title: Loss of FBXW7-mediated degradation of BRAF elicits resistance to BET inhibitors in adult T cell leukemia cells

doi: 10.1186/s12943-020-01254-x

Figure Lengend Snippet: FBXW7 wild type and mutants selectively target BRAF for proteasome degradation. a 293 T cells were co-transfected with FBXW7 and increasing amounts of BRAF for 48 h. FBXW7-mediated degradation of BRAF was then determined by Western blot. 293 T cells were co-transfected with FBXW7 and BRAF or BRAF V600E mutant for 48 h. FBXW7-mediated degradation of BRAF or BRAF V600E was determined by Western blot. b FBXW7-mediated ubiquitination of BRAF. 293 T cells were co-transfected with FBXW7, K48-Ub and BRAF. Cells were treated with MG132 for 6 h before harvest. Immunoprecipitated BRAF and Western blot Ub showed the ubiquitination level of BRAF. c Comparison of FBXW7 wild type and mutants W406R, W425R and S462P in their ability to target BRAF for degradation following transient transfection in 293 T cells. The interaction between FBXW7 WT/mutants and BRAF was analyzed by co-immunoprecipitation. 293 T cells co-transfected with wild-type or mutant FBXW7 and BRAF were lysed and immunoprecipitated with BRAF. Western blot was used to analyze the interaction between FBXW7 WT/mutants and BRAF. d ATL cells MT1 stably transfected with a TET-inducible FBXW7 wild type or W425R were induced with doxycycline and degradation (WT) or lack thereof (W425R) of endogenous BRAF was confirmed by Western blot. Endogenous degradation of BRAF by wild type FBXW7 was confirmed by immunohistochemistry following induction of FBXW7 with doxycycline. e Graphical representation showing the correlation between FBXW7 gene alternation in T-ALL cell lines and JQ1 IC 50. f Data from RPPA was used to quantify BRAF and c-MYC protein expression in MT1 cells expressing FBXW7 WT, R465C, R505C and R479Q. g FBXW7-mediated degradation of BRAF by wild type FBXW7 and R505C but not R465C was confirmed in transient transfection assays and Western blot analyses. h FBXW7 R505C retains wild type ability to add ubiquitin to BRAF. 293 T cells were co-transfected with FBXW7, K48-Ub and BRAF. Cells were treated with MG132 for 6 h before harvest. Immunoprecipitated BRAF and Western blot Ub showed the ubiquitination level of BRAF. i Combination therapy with Ulixertinib or PLX8394 prevents tumor cell resistance to BET inhibitors. MT1 cells carrying an inducible wild type FBXW7 or S462P mutant were cultured in presence of doxycycline and in the presence of JQ1 or JQ1 in combination with BRAF inhibitor PLX8394 or ERK inhibitor Ulixertinib. After 48 h hours cells were collected, and cellular proliferation measured by XTT assays. FBXW7 WT cells treated with JQ1 alone was set as 100% reference. j Following the same settings as in ( i ), cellular proliferation was measured by XTT assays every 2 days for 8 days and Doxycycline was added to the media every 2 days

Article Snippet: Endogenous expression of proteins was performed using antibodies for CyclinE (Santa-Cruz #sc-198), Mcl-1 (Santa-Cruz #sc-12,756), c-myc (9E10), FBXW7 (Proteintech #28424–1-AP), BRAF (Santa-cruz #sc-5284), p-MEK-1/2 (Calbiochem), p-ERK-1/2, and STAT3 (Santa-Cruz #sc-482).

Techniques: Transfection, Western Blot, Mutagenesis, Immunoprecipitation, Stable Transfection, Immunohistochemistry, Expressing, Cell Culture