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Sample GSM2067608 Query DataSets for GSM2067608
Status Public on Jun 01, 2016
Title TC32: OSI906-Selected-C5
Sample type protein
 
Source name Resistant Ewing Sarcoma Cell line to IGF-1R/IR blockade
Organism Homo sapiens
Characteristics cell type: Ewing Sarcoma cell line
cell line: TC32 clone
Treatment protocol TC32 and TC71 ES clones with acquired resistance to OSI-906 or NVP-ADW-742 were generated by maintaining the corresponding parental cell lines with increasing concentrations of the agents (up to 2.3 μM for OSI-906, 1.5 μM for NVP-ADW-742) for 7 months. All parental and acquired drug resistant cell lines were tested twice per year for mycoplasma contamination using the MycoAlert Detection Kit (Lonza Group Ltd.) according to the manufacturer’s protocol and validated using short-tandem repeat fingerprinting with an AmpFLSTR Identifier kit as previously described. Herein, we determine subtle differences in acquired mechanism of resistance by two promising small molecule inhibitors of IGF-1R/IR-α. OSI-906, which inhibits IGF-1R and IR, and NVP-ADW-742, which inhibits only IGF-1R, were evaluated using in vitro assays to decipher the mechanism(s) by which IGF-1R inhibition induces drug resistance in Ewing sarcoma cells.
Growth protocol Ewing Sarcoma cell lines sensitive or resistant to OSI-906 and NVP-ADW-742 grown in 2D-monolayer cultures were maintained in RPMI 1640 medium (Mediatech) containing 10% (vol/vol) fetal bovine serum (Gemini Bio-Products) and antibiotics (100 IU/ml penicillin and 100 mg/ml streptomycin [Mediatech]) in a humidified incubator at 37°C in a 5% CO2 atmosphere.
Extracted molecule protein
Extraction protocol Protein extraction from cell lines was performed by homogenizing an approximate 10 mg of frozen tissue in 500 ul of the lysis buffer containing protease and phosphatase inhibitors using an electric tissue homogenizer (Pro Scientific). Total lysed proteins were quantified using BCA protein assay kit and stored for further analyses for RPPA.
Label 115 Primary Antibodies
Label protocol Using a 2470 Arrayer (Aushon BioSystems), sample arrays were were processed, spotted onto nitrocellulose-coated FAST slides.
 
Hybridization protocol Protein-lysed-spots were probed with 179 validated primary antibodies and detected using a DakoCytomation-catalyzed system with secondary antibodies.
Scan protocol Slides were scanned on a flatbed scanner to produce 16-bit tiff images. Spots from tiff images were identified and their densities were quantified by MicroVigene. Relative protein levels for each sample were determined by interpolation of each dilution curve from the standard curve (supercurve) of the slide (antibody). Supercurve is constructed from a script in R written by the informatics department. These raw values are given as log2 values. MicroVigene software program (VigeneTech) was used for automated spot identification, background correction, and individual spot-intensity determination.
Description RPPA
Data processing Expression data was normalized for possible unequal protein loading, taking into account the signal intensity for each sample for all antibodies tested. Log2 values were media-centered by protein to account for variability in signal intensity by time and were calculated using the formula log2 signal – log2 median. Principal component analysis was used to check for a batch effect and feature-by-feature two-sample t-tests were used to assess differences between treatment and control groups. We also used feature-by-feature one-way analysis of variance (ANOVA) followed by the Tukey test to perform pair comparisons for all groups. Beta-uniform mixture models were used to fit the resulting p value distributions to adjust for multiple comparisons. The cutoff p values and number of significant proteins were computed for several different false discovery rates (FDRs). Biostatistical analyses comparing two groups were performed using an unpaired t-test with Gaussian distribution followed by the Welch correction. To distinguish between treatment groups, we used one-way ANOVA with the Geisser-Greenhouse correction. Differences with p values <0.05 were considered significant. Within clustered image maps (CIM), unsupervised double hierarchical clustering used the Pearson correlation distance and Ward’s linkage method as the clustering algorithm to link entities (proteins or genes) and samples.
 
Submission date Feb 19, 2016
Last update date Jun 01, 2016
Contact name Joseph A. Ludwig
E-mail(s) jaludwig@mdanderson.org
Phone 713-792-4265
Organization name UT-MD-Anderson Cancer Center
Department Sarcoma Medical Oncology
Lab Sarcoma Medical Oncology
Street address 4SCR2-1042 1901 East Rd
City Houston
State/province Texas
ZIP/Postal code 77054
Country USA
 
Platform ID GPL21488
Series (2)
GSE78121 In vitro proteomic expression changes in Ewing sarcoma cell lines after IGF-1R or IGF-1R/IR blockade
GSE78124 Insulin-Like Growth Factor Receptor 1 and Mammalian Target Of Rapamycin Blockade: Novel Mechanisms of Resistance and Synergistic Drug Combinations for Ewing Sarcoma

Data table header descriptions
ID_REF
VALUE Normalized signal intensity

Data table
ID_REF VALUE
4E-BP1-R-V -0.482301647
4E-BP1_pS65-R-V 1.797477573
53BP1-R-C -1.523077261
ACC_pS79-R-V 0.374296156
ACC1-R-C 1.021732302
Akt-R-V -0.478408162
Akt_pS473-R-V -0.68261667
Akt_pT308-R-V -0.090299125
alpha-Catenin-M-V 0.969303713
AMPK_alpha-R-C 1.11902948
AMPK_pT172-R-V 1.276516025
Annexin_I-R-V -0.478420574
AR-R-V -1.281975266
Bak-R-C 0.333583818
Bax-R-V -1.541531412
Bcl-2-M-V -0.957490005
Bcl-xL-R-V -1.300212607
Beclin-G-V 0.506782482
beta-Catenin-R-V 0.305853937
Bid-R-C -0.29974629

Total number of rows: 115

Table truncated, full table size 2 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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