Peptide Evidence

This compressed file contains a tab-delimited table listing the peptide evidence generated by processing the raw MS and MS/MS data using the MaxQuant software package, which includes a built-in database search engine called Andromeda. The spectra were searched against the UniProt Human Reference Proteome, accessed on August 2012. The table includes all instances of peptide identifications and quantitations, and their database search scores and posterior error probabilities

Figure 1

<p><b>A.</b> Mutant read frequency in different samples. The patient's blood samples were all taken around the time of neuroblastoma diagnosis. Numbers of sequencing reads per sample are displayed in the top level. * p = 0.01, comparing mutant read count frequencies between samples PD9058b3 and PD9058b with Fisher's exact test. Differences between PD9058b2 and sample b/b3 are not significant (p>0.05). <b>B.</b> Capillary sequencing of control germline DNA (Reference sequence) and patient's blood samples extracted at three time points, showing very low level of adenine (black arrow). The guanine (black peak) dropped approximately between 8–17% relative to control, as given by the software, which would not usually be classed as significant when looking for heterozygous germline mutations. <b>C and D.</b> Immunohistochemistry to show strong nuclear staining p53 in neuroblastoma (C) and sarcoma NOS (D).</p

EWSCs are sensitive to PARP1 trapping.

<p><b>(A)</b> Relative viability of mock-transfected and PARP1 siRNA-transfected ES8 cells treated with vehicle or olaparib. Asterisks indicate <i>student’s paired t-test P</i> value **P<0.01, ns = not significant. <b>(B)</b> PARP1 expression in cells transfected with a scrambled control or a titration of PARP1_1 siRNA and their relative viability following treatment with vehicle or olaparib. <b>(C)</b> IC50 values of parental ES8 and PARPi-resistant OLAR5 cells to five different PARPi and the fold difference between them. <b>(D)</b> Western blot of PARP1 expression in ES8 and OLAR5 cells. Viability values are the mean of technical triplicates and representative of 3 independent experiments.</p

EWSCs are sensitive to PARP inhibition and S-phase DNA-damaging agents.

<p><b>(A)</b> and <b>(C)</b> Scatter plots of IC₅₀ (μM) values on a log scale comparing drug sensitivity of <i>EWS-FLI1</i>-positive and wild-type (WT) <i>EWS-FLI1</i>-negative cell lines to (A) four PARPi and (C) camptothecin and cisplatin. The sample size (n) is indicated and each circle represents the IC₅₀ of one cell line. The red bar is the geometric mean and the drug name is depicted above each plot along with the significance of the association as determined by an unpaired two-sample t-test. <b>(B)</b> Long term viability assays in EWSCs were performed in the presence of vehicle (-) or increasing concentrations of four PARPi as indicated. Non-EWSC lines (U-2-OS and HeLaSF) are included for comparison. These data are representative of 3 independent experiments.</p

Temozolomide enhances PARP inhibitor sensitivity in multiple tumour types.

<p>List of cell lines screened against a combination of olaparib and temozolomide. Whether enhancement of PARP inhibitor sensitivity with temozolomide is observed (✔) or not (✖) is indicated.</p

DNA DSB repair by HR is functional in EWSCs.

<p><b>(A)</b> Western blot of ES8 cells treated with olaparib for the times indicated. Markers are grouped as part of ATM or ATR signaling. Tubulin served as a loading control. (<b>B)</b> Western blot of ES8 cells treated with camptothecin and harvested at various time points following drug washout. GAPDH served as a loading control. <b>(C)</b> Percentage of EdU-positive and EdU-negative ES8 cells with >5 nuclear RAD51 foci following 6-hour treatment with vehicle or olaparib (ola). <b>(D)</b> Olaparib log GI₅₀ (μM) of cell lines mock-transfected or transfected with CtIP or BRCA1 siRNA as indicated.</p