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