*4.2. Cell Line and Cell Culture*

The NSTS-47 cell line was established in our laboratory with the procedure previously described [30]. A tumor sample was obtained from the same boy mentioned in the previous paragraph during curative surgical procedure when he was 1 year and 7 months old. Cells were grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 20% fetal calf serum (FCS), 2 mM glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin (all purchased from GE Healthcare Europe GmbH, Freiburg, Germany). The cell line was maintained under standard conditions at 37 ◦C in a humidified atmosphere containing 5% CO2 and subcultured one or two times per week. Cells from passage number 8 to 19 were used for experiments.

#### *4.3. Genetic Analyses*

The mutation in *PDGFRB* was identified by Sanger sequencing using an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and confirmed by whole exome sequencing (WES). In all cases, WES was performed using the TruSeq Exome Kit, NextSeq® 500/550 Mid Output Kit v2 and NextSeq 500 (all Illumina, San Diego, CA, USA).

#### *4.4. Chemicals*

Sunitinib, erlotinib, U0126 (all purchased from Cell Signaling Technology, Danvers, MA, USA) and FR180204 (Sigma-Aldrich, St. Louis, MO, USA) were prepared as a 20 mM stock solution in dimethyl sulfoxide (DMSO) and stored at −20 ◦C. PDGF-BB (Cell Signaling Technology) was prepared at a concentration of 100 μg/mL in 20 mM citric acid (pH 3.0) supplemented with 0.8% BSA (bovine serum albumin) and stored at 4 ◦C. EGF (Sigma-Aldrich) was prepared at a concentration of 100 μg/mL in 10 mM HCl and stored at 4 ◦C. For the determination of proliferative activity, concentrations of protein kinase inhibitors (PKIs) ranging from 0.001 to 10 μM and PDGF-BB concentrations of 0.25 and 10 ng/mL were tested. For Western blot analyses, PKI concentrations ranging from 0.05 to 10 μM, PDGF-BB concentrations of 10 and 30 ng/mL and EGF concentrations of 40 and 100 ng/mL were used.

#### *4.5. Phospho-RTK and Phospho-MAPK Array Analysis*

The relative phosphorylation levels of 49 RTKs were analyzed using the Human Phospho-RTK Array kit (R&D Systems, Minneapolis, MN, USA), and the relative phosphorylation levels of 26 proteins, including 9 MAPKs, were determined using the Human Phospho-MAPK Array kit (R&D Systems) according to the manufacturer's protocol. The levels of phosphorylation were quantified using ImageJ software [31] and normalized to control spots and the background. The analysis was performed as described in previous studies [8,32].

#### *4.6. MTT Assay*

The MTT assay was used to determine the proliferative activity of the NSTS-47 cell line. A total of 103 cells were seeded in 200 μL of culture medium into each well of 96-well microplates, and cells were allowed to adhere overnight. The next day, the medium was carefully removed, and fresh medium containing various concentrations of chemicals described above or control medium was added. The microplates were incubated under standard conditions. To evaluate changes in cell proliferation, the medium was removed and replaced with 200 μL of fresh DMEM containing 3-(4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at a concentration of 0.5 mg per mL. The microplates were then incubated at 37 ◦C for 3.5 h. The medium was carefully removed, and the formazan crystals were dissolved in 200 μL of DMSO. The absorbance was measured at 570 nm using a Sunrise Absorbance Reader (Tecan, Männedorf, Switzerland), with a reference absorbance at 620 nm.
