Fig 1: RNASE7 and NELL2 do not function as ROS1 ligands. (a) Immunoblot analysis of total ROS1 levels using the D4D6 anti-ROS1 antibody in lysates made from HEP3B, LN-428, MCF10A and TONG cell lines. (b) Comparison of ROS1 mRNA expression level in hepatocellular carcinoma cell lines compared to positive control samples. mRNA Z-score relative to 1156 cancer cell lines in the CCLE database. (c) Immunoblotting of lysates generated from SAOS2, U20S, SW-1353, MCF10A parental, MCF10A CDH1−/−, Hs578, and BT-549 cell lines with the D4D6 and OTI1A1 anti-ROS1 antibodies to detect endogenous ROS1, and anti-β-Actin antibody for determining protein loading. (c) Endogenous ROS1 immunopreciates generated using the D4D6 antibody, from indicated cell lines, were probed with phospho-ROS1 and total ROS1 antibodies as shown. (d, e) Immunoblotting with phospho- and total ROS1, SHP2 and ERK1/2 antibodies from SW-1353 cells after treatment with recombinant RNASE7 (e) or NELL2 (f) as indicated. (g) Coommassie stained gel shows fidelity of recombinant RNASE7 and NELL2; 500 ng of these proteins is compared to 500 ng of bovine serum albumin as a reference control.
Fig 2: ROS1 extracellular domain deletions identify key regulatory regions. (a) Illustration of the engineered ROS1 extracellular domain deletion mutant. White vertical line indicates signal peptide, 1–9 denote FNIII domains, ß1–ß3 denote ß-propellers, black vertical line indicates transmembrane domain, and TKD indicates tyrosine kinase domain. (b) Representative immunoblot analysis of phosphorylated and total ROS1 and ERK in cell lysates prepared from HEK293T17 cells transfected with either ROS1 wildtype (WT) or N-terminal truncation constructs. Representative images from three independent experiments are shown. (c) Densitometry of phosphorylated ROS1 relative to total ROS1 for the transfected constructs (pixel density of phospho-ROS1 Y2274/pixel density of total ROS1). Data shown from three independent replicates. (d) Immunoblot analysis of phosphorylated and total ROS1 and ERK from HEK293A cell lysates transfected with ROS1 WT, indicated ECD deletion constructs and CD74::ROS1, EZR::ROS1 and SLC34A2 (SLC)::ROS1 oncofusions. Representative images from two independent experiments are shown.
Fig 3: Interaction of ROS1 ECD deletion constructs with self and effector protein SHP2. (a) Immunoblot analysis of total ROS1, phospho-ROS1 and SHP2 protein levels from ROS1-Myc-immunoprecipites generated from transiently transfected HEK293A cells, as indicated. The Myc epitope is engineered on the carboxy-terminus of ROS1. (b–d) Immunoblot analysis of Flag- or Myc-tagged ROS1 variants, as indicated, in Flag immunoprecipitates and input lysates generated from HEK293A cells co-transfected with indicated combinations of Myc- and Flag-ROS1 constructs.
Fig 4: Deletion of the ROS1 extracellular domain, including the sixth fibronectin III domain, is sufficient for oncogenic transformation. (a) Illustration comparing ΔFN1-8 and CD74::ROS1 domain organization. Grey line indicates signal peptide, black lines show FNIII 9, red line indicates transmembrane domain, TKD indicates tyrosine kinase domain, and teal oval indicates CD74 portion of CD74::ROS1 fusion. (b) Immunoblot analysis of phosphorylated and total proteins in cell lysates generated from NIH3T3 ROS1 WT, truncation, and CD74::ROS1 cell lines. (c) Densitometry of relative phosphorylated ROS1 normalized to total ROS1 (pixel density of phospho-ROS1 Y2274/pixel density of total ROS1). Data shown from three independent experiments are shown. (d) Representative images from anchorage-independent soft agar assays using NIH3T3 ROS1 WT, truncation, and CD74::ROS1 cell lines. (e) Fold change in colony number of ROS1 truncation mutants relative to average ROS1 WT colony number. Data are from sixteen wells per cell line. Ordinary one-way ANOVA test was used to determine statistically significant differences with p < 0.05 indicating significance. (f) IL-3 withdrawal assay for Ba/F3 cells transduced with ROS1 truncations and CD74::ROS1. Total viable cell number was determined by counting cells every other day after IL-3 withdrawal. Fold change is displayed relative to day 0 for each cell line. (g) Heat map of IC50 values (nanomolar) of crizotinib, entrectinib, lorlatinib, repotrectinib, taletrectinib, and cabozantinib for Ba/F3 ROS1 truncation and CD74::ROS1 cell lines. IC50 values were calculated from triplicate dose response curves. Color scale for heatmap is indicated in the figure.
Supplier Page from DNASU for ROS1 (Homo sapiens) in pENTR223.1 (Gateway donor/master vector)