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Page 4 of 11 Fonseka et al. J Cancer Metastasis Treat 2020;6:7 I http://dx.doi.org/10.20517/2394-4722.2019.024
TM
(Invitrogen ) at 20 V for 7 min. The membrane was incubated with blocking solution containing 5% (w/v)
skim milk in Tris-buffered saline with 0.05% Tween 20 (TTBS) [100 mM Tris-HCl pH 7.5, 150 mM NaCl
and 0.05% (v/v) Tween 20] for 45 min. The membrane was washed three times with TTBS (10 min each)
and probed with the relevant primary antibody overnight at 4 °C. The membrane was again washed with
TTBS over 30 min. Subsequently, the blot was probed with appropriate IRDye (LI-COR®) or peroxidase
(Sigma-Aldrich®) conjugated secondary antibody for 1 h at room temperature. The blot was then washed
three times with TTBS over 30 min. For the visualisation of the protein bands probed with IRDye (LI-COR®),
ODYSSEY CLx (LI-COR®) machine was used.

Cell death assay
3
Cells (5 × 10 per well) were seeded in a 24-well plate in 500 µL DMEM medium and allowed to adhere
for 48 h at 37 °C in the presence of 5% CO . Cells were then treated with or without doxorubicin (1 µM)
2
and incubated for 48 h. For combinational treatment studies, curcumin (10 µM) or silibinin (100 µM)
treatment was performed 24 h before the addition of doxorubicin (pre-treatment) as well as on the same
day of doxorubicin treatment (combinational). After 48 h of cancer therapeutic drug treatment, cells were
scraped and resuspended. Supernatant (300 µL) was then transferred in to a 96-well plate and spun at
300 g for 5 min before discarding the supernatant. The remaining pellet was then resuspended with 200
µL of propidium iodide (PI) buffer [0.1% (w/v) TritonX 100 and 50 µg/mL Propidium iodide (Sigma Life
4
Science®] and was incubated overnight at 4 °C. For this, 2 × 10 cells were used in the analysis, Results from
fluorescence activated cell sorting CANTO II (BD Biosciences) were then analysed using FlowJo (TreeStar).

Establishment of tumour xenografts
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SK-N-BE2 cells (5 × 10 ) were subcutaneously injected to athymic Balb/c nude female mice (8 weeks old). The
cells were suspended in Matrigel before the injections. After formation of tumours, the mice were injected
intraperitoneally (i.p.) with dimethyl sulfoxide (DMSO) (control), curcumin (40 mg/kg), doxorubicin (5 mg/
kg) and combinational treatment of doxorubicin and curcumin three times a week. Tumour size and the
2
weight of the mice were measured daily. The tumour volume was calculated according to the formula ½(W × L).
According to the Australian code of practice for the care and use of animals for scientific purposes and La Trobe
3
Ethics Committee guidelines (AEC 14-15), mice were sacrificed when the tumour size reached 1500 mm .
Statistical analysis
Statistical significance of experiments was analysed by student t-test and P values less than 0.05 were
considered to be statistically significant. CI (Combination index) was calculated using Chou-Talalay
[41]
method . A violin plot was generated using MATLAB and the P value of the violin plot was also
[42]
calculated using MATLAB .

RESULTS
Neuroblastoma cells exhibit mesenchymal signature
Prior to commencement of the experiments, publicly available RNA-Seq data for neuroblastoma tissues
and cells were queried for the expression of EMT genes to examine the hypothesis of whether the
neuroblastoma cells are mesenchymal. To validate the expression of epithelial and mesenchymal genes,
publicly available RNA-Seq data for neuroblastoma cell lines and tissues were examined. In total, 315 genes
[43]
implicated in EMT were retrieved from the public literature and the expression profile of these genes
were plotted in a panel of neuroblastoma cell lines and tissues [Figure 1]. Interestingly, agreeing with our
hypothesis, neuroblastoma cell lines (n = 8) and tissues (n = 157) exhibited a high mesenchymal and low
epithelial gene expression. These results suggest that neuroblastoma cells are mesenchymal and hence could
contribute to the aggressive phenotype.