Cell lines and tumors
BaF3 and Karpas-299 cells were obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany). U118 MG, HCC78, and 3T3 cells were purchased from American Type Culture Collection (Manassas, VA). BaF3 cells were maintained in RPMI-1640 medium (Invitrogen) with 10% fetal bovine serum (FBS) (Sigma) and 1.0 ng/ml IL-3 (R&D Systems). Karpas-299 cells were grown in RPMI-1640 with 10% FBS. Other cell lines were grown in DMEM with 10% FBS.
Cholangiocarcinoma tumors (n
23), as well as matching para-tumor tissues (n
20) were collected within 15 minutes from surgical resections from patients when sufficient material for PhosphoScan® analysis, RNA, and DNA extractions were available. According to the Edmondson grading system, all tumor samples have differentiation grades II–III. The tumor specimens were collected at RuiJin hospital (Shanghai, China) and Third Xiangya hospital (Changsha, Hunan, China) with written consent from patients. Patient information was not revealed in this study, and the data were analyzed anonymously. Obtaining patient materials were approved by both Ruijin hospital and third Xiangya hospital institutional review board.
Phosphopeptide immunoprecipitation and analysis by LC-MS/MS Mass Spectrometry
Phosphopeptides were prepared using PhosphoScan® Kit (Cell Signaling Technology). In brief, about 200–500 mg tumor samples were homogenized and lysed in urea buffer, trypsin digested lysates were purified by Sep-pak C18
column (Waters). Then, lyophilized peptides were redissolved and immunoaffinity purified with pY-100 antibody. pTyr-containing peptides were concentrated on reverse-phase micro tips. LC-MS/MS analysis was performed with an LTQ Orbitrap Mass Spectrometer (Thermo Fisher Scientific) and a peptide mass accuracy of ±3 ppm was one of the filters used for peptide identification. Details were described previously 
. In brief, samples were collected with an LTQ – Orbitrap hybrid mass spectrometer, using a top-ten method, a dynamic exclusion repeat count of 1, and a repeat duration of 30 sec. MS spectra were collected in the Orbitrap component of the mass spectrometer and MS/MS spectra was collected in the LTQ. Sequest (Thermo Fisher Scientific) searches were done against the NCBI human database released on July 02, 2009, (containing 37,391 proteins), allowing for tyrosine phosphorylation (Y+80) and oxidized methionine (M+16) as differential modifications. The PeptideProphet probability threshold was chosen to give a false positive rate of 5% for the peptide identifications
For each patient sample, each protein's spectral counts were normalized to those for GSK3A (100). We used the following statistical and computational tools from GenePattern 3.0 software package (Broad Institute of MIT and Harvard) for Comparative Marker Selection; from MultiExperiment Viewer version 4.4 for Hierarchical Clustering (Pearson correlation distance and complete linkage clustering).
Rapid Amplification of Complementary DNA Ends
RNeasy Mini Kit (Qiagen) was used to extract RNA from human tumor samples. DNA was extracted with the use of DNeasy Tissue Kit (Qiagen). Rapid amplification of cDNA ends was performed with the use of 5′ RACE system (Invitrogen) with primers ROS-GSP1 for cDNA synthesis and ROS-GSP2 and ROS-GSP3.1 for a nested PCR reaction, followed by cloning and sequencing PCR products.
Transfection, cell proliferation and growth assays
Transfections were carried out using FuGENE 6 (Roche Diagnostics), and retrovirus was harvested at 48 after transfection. BaF3 cells were transduced with retroviral supernatant containing either the MSCV-Neo/FIG-ROS(L) or MSCV-Neo/FIG-ROS(S) vector, and selected for G418 (0.8 mg/ml). IL-3 independent growth was accessed by plating transduced BaF3 cells in IL-3 free medium, after the cells were washed three times in PBS. For dose response curves, cells were incubated for 72 hours in the presence of TAE684 (customer synthesized), and the number of viable cells was determined with the CellTiter 96 AQueous One solution cell proliferation assay (Promega). IC50 was calculated with the use of OriginPro 6.1 software (OriginLab). The percentage of apoptotic cells at 48 hours was determined by flow cytometric analysis of cleaved caspase-3 (Cell Signaling Technology).
3T3 cells stably transfected with myc tagged FIG-ROS(L), FIG-ROS(S), or empty vector were subjected to immunofluorescence assay according to protocol (Cell Signaling Technology).
For RT-PCR, first-strand cDNA was synthesized from 2.5 ug of total RNA with the use of SuperScript™ III first-strand synthesis system (Invitrogen) with oligo (dT)20. Then, the FIG-ROS fusion gene was amplified with the use of primer pairs FIG-F2 and ROS-GSP3.1. Wild type FIG and ROS gene was amplified with the use of primer pairs FIG-F3 and FIG-R8, ROS-Ex31F and ROS-GSP2, respectively. For genomic PCR, amplification of the fusion gene was performed with the use of LongRange PCR kit (Qiagen) with primer pairs FIG-F3 and ROS-GSP3.1 for TC23, or FIG-F7 and ROS-GSP4.1 for TC03 and U118MG.
The following primers were used:
FIG-F7: 5′ TGTGGCTCCTGAAGTGGATTCTGA
The open reading frame of the FIG-ROS(L) and FIG-ROS(S) fusion gene was amplified by PCR from cDNA of ROS fusion positive patient tumors. These PCR products were cloned into the retroviral vector MSCV-Neo with a C-terminal Myc tag.
Cells were lysed in 1× cell lysis buffer (Cell Signaling Technology) supplemented with Protease Arrest™ (G Biosciences) and separated by electrophoresis. All antibodies and reagents for immunoblotting were from Cell Signaling Technology.
Soft agar assay and Xenograft
Retroviral transduced 3T3 cells were selected for G418 (0.5 mg/ml) for 7 days, and the cells were then cultured in soft agar in triplicate for 17 days. 1×106 transduced 3T3 cells were resuspended in Matrigel (BD Biosciences) and injected subcutaneously at 2 sites into each nude mice. Each cell line was tested in 4 mice with a total of 8 injections. Mice were monitored daily for tumor formation and size, and were sacrificed when tumors reached approximately 1 cm×1 cm.
Approval for the use of animals in this study was granted by Cell Signaling Technology Animal Care and Use Committee with approval ID 650.
In vitro kinase assay
Cell lysates from FIG-ROS transfected BaF3 cells were subjected to immunoprecipitation with Myc-Tag antibody, ROS immune complex were washed 3 times with cell lysis buffer, followed by kinase buffer (Cell Signaling Technology). Kinase reactions were initiated by re-suspending the ROS immune complex into 25 ul kinase buffer that contains 50 uM ATP, 0.2 uCi/ul [gamma32p] ATP, with 1 mg/ml of Poly (EY, 4
1). Reactions were stopped by spotting reaction cocktail onto p81 filter papers. Samples were then washed and assayed for kinase activity by detection with a scintillation counter.