Fig. S1 Related to Figure 1

A. The expression of the other components of CPC in different NB cell lines was

detected by western blot (from the same experiment for Fig.1A). B. The expression

of the other components of CPC during RA-induced NB differentiation (day 0, 2, 4,

and 6) was detected by western blot. C. Left panel: INCENP mRNA levels in different

stage NB tumors (Kocak-649 dataset). Right panel: INCENP mRNA levels in MYCN-

WT and MYCN-amp NB tumors (all stages, Kocak-649 dataset). D. MYCN and RNA

pol II binding sites at the promoter regions of INCENP, BIRC5 (Survivin), CDCA8

(Borealin) and AURKB (Aurora B) in BE2C cells were analyzed based on ChIP-seq

data from GSM2113521 and GSM2113522 datasets using R2 platform (r2.amc.nl/ ) .

E. Left panel: western blot analysis of the expression of INCENP, Aurora B, Survivin,

Borealin and MYCN in Tet21N cells with or without Tetracycline (Tet, 1 μg/mL)

treatment for 48 hr. Right panel: western blot analysis of the expression of INCENP,

Aurora B, Survivin, Borealin and MYCN in MYCN knockdown BE2C cells. F and G.

Event-free (F) and overall (G) Kaplan-Meier plots based on the expression of

INCENP in tumors from NB patients at all stages. All the data are from Kocak-649

dataset in which only 476 patients have survival data. H and I. Event-free (H) and

overall (I) Kaplan-Meier plots based on the expression of INCENP in tumors from

high-risk NB patients including stage 4 patients > 18 months (upper panel) and

patients whose tumors harbor MYCN amplification (lower panel) in Kocak-649

dataset in which only 476 patients have survival data.

Fig. S2 Related to Figure 2

A. A schematic diagram of the design of siRNA resistant form of INCENP. B and C.

Rescue experiment was performed in INCENP depleted BE2C cells by

overexpression of siRNA resistant form of INCENP. B, The results of confluence

analysis from Incucyte machine. The values of cell confluence at endpoint (90 hr) is

used for statistical analysis by standard two-tailed student’s t test (p1<0.001: siCTRL

dox+ vs. siINCENP-#2 dox-; p2<0.001: siINCENP-#2 dox+ vs. siINCENP-#2 dox-;

p3=0.49: siINCENP-#2 dox+ vs. siCTRL dox+). Bars show the average of three

replicates ± SD. C, Cell number counting at day 3 after siRNA transfection and

INCENP overexpression upon dox treatment. Data are represented as mean SD of

triplicates. D. INCENP knockdown in KCNR cells. Left panel: cell confluence from

Incucyte machine. The values of cell confluence at endpoint (168 hr) is used for

statistical analysis by standard two-tailed student’s t test; Right panel: q-PCR

analysis of INCENP knockdown efficiency. Bars show the average of three replicates

± SD. E. INCENP knockdown in Kelly cells. Left panel: cell confluence from Incucyte

machine. The values of cell confluence at endpoint (162 hr) is used for statistical

analysis by standard two-tailed student’s t test; Right panel: q-PCR analysis of

INCENP knockdown efficiency. Bars show the average of three replicates ± SD. F.

Heat map of growth dependency of all the four CPC components in different NB cell

lines (data from Project Achilles Data Portal and heat map was generated in

Rstudio).

Fig. S3 Related to Figure 3

A. The confluence analysis of BE2C, NGP and SY5Y inducible shCTRL cells treated

with doxycycline (dox) or vehicle in Incucyte machine. Western blot analysis of

INCENP knockdown level in these control cells after 72-hr treatment with dox are

also shown. The values of cell confluence at 120 hr is used for statistical analysis by

standard two-tailed student’s t test. Bars show the mean ± SD of three replicates. B.

Adherent colony formation ability of BE2C, NGP and SY5Y inducible shCTRL cells

was determined by colony formation assay. The relative number of colonies (upper

panel) and the representative pictures for each cell type after crystal violet staining

are shown (lower panel). Bars show the average of three replicates ± SD. C. The

tumorigenesis ability of BE2C, NGP and SY5Y inducible shCTRL cells was

determined by soft agar assay in vitro. The relative colony numbers (upper panel)

and the representative pictures of colonies grown in soft agar for each cell type are

shown (lower panel). Data represent mean SD of three replicates.

Fig. S4 Related to Figure 4

-

A. BE2C-shCTRL xenograft tumor growth in mice treated with doxycycline (dox)-

containing (n=10) or control chow (n=10). Upper panel: xenograft growth curve by

average tumor volume of each group; Lower panel: xenograft growth curve by tumor

volume of individual mouse in each group. Bars show the tumor size average of the

mice/group SEM. B. SY5Y-shCTRL xenograft tumor growth in mice treated with

doxycycline (dox)-containing (n=6) or control chow (n=7). Upper panel: xenograft

growth curve by average tumor volume of each group; Lower panel: xenograft growth

curve by tumor volume of individual mouse in each group. Bars show the tumor size

average of the mice/group SEM. C and D. Kaplan-Meier graphs showing the

murine survival of BE2C-shCTRL (C) and SY5Y-shCTRL (D) tumor-bearing mice

upon dox treatment. The statistical significance between two treatment groups was

evaluated using a log rank test.

Fig. S5 Related to Figure 5

A. The results of immunostaining (DAPI: Cyan; a-Tubulin: Magenta) showing the

irregular nuclear shape of INCENP depleted SY5Y cells at 48 hr post transfection.

Scale bars, 20 m. B. H&E staining results of SY5Y-shINCENP xenografts without

(upper panel) or with (lower panel) dox treatment. Scale bars, 50 m. C. Cell cycle

analysis of SY5Y cells transfected with siCTRL, siINCENP-#2 and siINCENP-#4 for

48 hr. Data represent mean ± SD of three replicates. D. The results of

immunostaining (DAPI: Cyan; a-Tubulin: Magenta) showing the multinucleated cells

in INCENP depleted 293T, ARPE19 and HeLa cells at 48 hr post transfection. Scale

bars, 20 m.

Fig. S6 Related to Figure 6

A. Heatmap showing up-and downregulated genes following 24 hr of INCENP

silencing in BE2C cells. Data are presented as normalized expression values of three

biological replicates based on Partek Flow software analysis and fold change (FC)

>=1.5, FDR <= 0.05 and p-value <= 0.05. B. Top ten differentially expressed

canonical pathways upon INCENP knockdown defined by Ingenuity Pathway

Analysis (IPA, QIAGEN). C. Western blot analysis of DNA damage markers-pH2A.X

and pCHK2 in BE2C, NGP and SY5Y shCTRL and shINCENP cells upon dox

treatment. D. Caspase-3/7 activity was detected in BE2C shCTRL or shINCENP

(left), NGP shCTRL or shINCENP (middle) and SY5Y shCTRL or shINCENP (right)

cells after 3 day of dox treatment. Data represent mean SD of three replicates. E.

qRT-PCR analysis showing relative mRNA levels of p21 in BE2C cells after INCENP

knockdown for 48 hr. Data represent mean SD of three replicates. F. Western blot

analysis of p21 expression upon INCENP and p53 double knockdown in BE2C cells.

G. Pan-Caspase inhibitor rescue experiment was performed in INCENP silenced

BE2C cells. DMSO or 20 M Z-VAD-fmk (Z-VAD) was applied to BE2C cells when

they were plated into multi-well plates right away after siRNA nucleofection. MTS

assay (left panel) was conducted to determine relative viable cell number

respectively after 72 hr Z-VAD treatment. Western blot analysis of the levels of

pH2A.X, cleaved Caspase-3 and cleaved PARP-1 in INCENP depleted BE2C cells

treated with Z-VAD for 72 hr (right panel). Data represent mean SD of three

replicates. H. Caspase-3/7 activity assay showing no apoptosis phenotype in

INCENP silenced 293T, ARPE19 and HeLa cells. Data represent mean SD of three

replicates.

Fig. S7 Related to Figure 7

A. β-gal staining of BE2C cells treated with DMSO or 250 nM or 500 nM Barasertib

(a specific Aurora B inhibitor) for 7 days. Bright field pictures (left panel) and

quantification data (right panel) are shown. Data represent mean SD of three

replicates. Scale bars, 50 m. B and C. β-gal staining of BE2C shINCENP (B) and

NGP shINCENP cells (C) following dox treatment for 10 days. Bright field pictures

(left panel) and quantification data (right panel) are shown. Data represent mean

SD of three replicates. Scale bars, 50 m.