Supplementary Information

Therapeutic targeting of YY1/MZF1 axis by MZF1-uPEP inhibits aerobic glycolysis and neuroblastoma progression

Fang et al.

GSE16476 GSE16476 GSE16476 GSE16476 GSE16476 GSE16476 P=2.3×10-3 10 P=6.0×10-4 P=2.8×10-3 P=4.0×10-4 13 P=1.3×10-6 P=9.9×10-3 12 8 11

10 6 9 log2 ENO1 intensity ENO1 log2 intensity log2 ALDOC 4 8 ≤18 ＞18 No Yes 2 4 ≤18 ＞18 No Yes 2 4 (n=48) (n=40) (n=55) (n=33) (n=15) (n=40) (n=48) (n=40) (n=55) (n=33) (n=15) (n=40) Age (months) Death INSS stage Age (months) Death INSS stage

GSE16476 GSE16476 GSE16476 GSE16476 GSE16476 GSE16476 P=1.0×10-5 P=1.1×10-5 P=2.6×10-3 12 P=7.9×10-7 P=1.8×10-10 P=2.3×10-4

8 4

log2 HK2 intensity HK2 log2 log2 GPIintensity 0 ≤18 ＞18 No Yes 2 4 ≤18 ＞18 No Yes 2 4 (n=48) (n=40) (n=55) (n=33) (n=15) (n=40) (n=48) (n=40) (n=55) (n=33) (n=15) (n=40) Age (months) Death INSS stage Age (months) Death INSS stage

GSE16476 GSE16476 GSE16476 GSE16476 GSE16476 GSE16476 13 -7 13 -7 -5 -4 × -8 P=1.1×10-5 P=1.2×10 P=9.7×10 P=1.5×10 P=2.0×10 P=4.9 10 12 12 11 11 10 10 log2 LDHA intensity LDHA log2 log2 intensityPGK1 9 9 ≤18 ＞18 No Yes 2 4 ≤18 ＞18 No Yes 2 4 (n=48) (n=40) (n=55) (n=33) (n=15) (n=40) (n=48) (n=40) (n=55) (n=33) (n=15) (n=40) Age (months) Death INSS stage Age (months) Death INSS stage

Figure S1. Expression of glycolytic in a public NB dataset. Mining of a public microarray dataset (GSE16476) revealing the levels of ALDOC, ENO1, GPI, HK2, PGK1, or LDHA in NB tissues with different status of age, death, or INSS stages. Student’s t test compared the difference. Bars are means and whiskers (min to max).

A C D MZF1 (GSE62564) SK-N-AS SK-N-AS P=5.8×10-3

100 ) Low (n=293) 80 ** ** ratio * 60 ** * High (n=205)

40 Renilla (qRT-PCR) 20 survival Percent ΔΔ ΔΔ Firefly/

0 ( 0 100 200 300 Relative transcript levels t t 1 activity luciferase Relative T u OC Months GPI HK2 GK W Mu MZF1 ENO1 LDHA P 1 ALD HK2 HK2 M B PGK1 PGKWT IMR-32 IMR-32

6 )

ratio 4 * *

2 * * Renilla (qRT-PCR) * ** Δ Δ levels (qRT-PCR) ** * ** ** Firefly/ 0 ( Relative MZF1 transcript Relative transcript levels

activity luciferase Relative t t G A 3 -C 2 1 u D 29 AS ) F1 K Mu WT M K N- Z GPI H 2 1 1 -SY5Y- M ENO1 LDHA PGK K K BE(2 IMR-32 HK2 WT MCF 10HE S ALDOC H SH PGK PGK

E HK2 HK2 F (GSE16476) (GSE62564) GSE16476 GSE16476 P=3.0×10-10 P=2.4×10-16 10 13 100 R=0.498 R=0.408 P=7.9 × 10-7 P=8.0 × 10-5 8 80 Low (n=350) n=88 12 n=88 Low (n=60) 60 6 11 40 High (n=148) 4 10 20 2 Log2 HK2 intensity Percent survival Log2 PGK1intensity Log2

Percent Percent survival High (n=28) 0 0 9 0 100 200 300 6 7 8 9 10 6 7 8 9 10 Months Months Log2 MZF1 intensity Log2 MZF1 intensity

PGK1 PGK1 (GSE16476) (GSE62564) P=5.3×10-8 P=3.0×10-23 100 80 Low (n=300) Low (n=51) 60 40 High (n=198) High (n=37) 20 Percent survival Percent Percent survival 0 0 100 200 300 Months Months

Figure S2. Roles of MZF1 and downstream glycolytic genes in NB. (A) Kaplan-Meier curves indicating the overall survival of 498 NB cases (GSE62564) with low or high levels of MZF1 (cutoff value=35.1). (B) Real-time qRT-PCR assay showing the transcript levels of MZF1 in normal dorsal root ganglia (DG), non-transformed MCF 10A cells, transformed normal HEK293 cells, and NB cell lines (normalized to -actin, n=4). (C) Real-time qRT-PCR assay (normalized to β-actin, n=4) indicating the transcript levels of MZF1, ALDOC, ENO1, GPI, HK2, LDHA, and PGK1 in SK-N-AS and IMR-32 cells stably transfected with empty vector (mock), MZF1, scramble shRNA (sh-Scb), or sh-MZF1. (D) Dual-luciferase assay showing the promoter activity of HK2 and PGK1 with wild-type (WT) or mutant (Mut) MZF1 binding site in SK-N-AS and IMR-32 cells stably transfected with mock, MZF1, sh-Scb, or sh-MZF1 (n=6). (E) Kaplan-Meier curves indicating the overall survival of 88 (GSE16476) and 498 (GSE62564) NB cases with low or high levels of HK2 (cutoff values=90.6 and 20.4) or PGK1 (cutoff values=1735.5 and 98.6). (F) The expression correlation of MZF1 with HK2 or PGK1 in 88 NB cases (GSE16476). Log-rank test for survival comparison in A and E. ANOVA and Student’s t test compared the difference in B-D. Pearson’s correlation coefficient analysis for expression in F. * P<0.05, ** P<0.01 vs. DG, mock, or sh-Scb. Δ P<0.05 vs. WT. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in B-D.

A Breast cancer B Endometrial carcinoma C Glioma D Head and neck carcinoma (TCGA) (TCGA) (TCGA) (TCGA) MZF1 MZF1 MZF1 MZF1 P=3.2×10-2 P=1.6×10-3 P=8.6×10-5 P=1.2×10-3 Low (n=141) Low (n=408) Low (n=593) Low (n=174) High (n=554) High (n=106)

survivalPercent Percent survival Percent Percent survival Percent Percent survival Percent High (n=66) High (n=109)

Months Months Months Months HK2 HK2 HK2 HK2 P=3.4×10-2 P=6.9×10-3 P=1.6×10-11 P=3.3×10-2 Low (n=123) Low (n=779) Low (n=332) Low (n=35) High (n=124) High (n=248) Percent survival Percent survival Percent Percent survival Percent High (n=183) survival Percent High (n=327)

Months Months Months Months PGK1 PGK1 PGK1 PGK1 P=3.8×10-3 P=4.9×10-3 P=2.2×10-17 P=1.0×10-3

Low (n=757) Low (n=222) Low (n=349) Low (n=195)

High (n=25) High (n=205) Percent survival Percent Percent survival Percent Percent survival Percent High (n=310) Percent survival High (n=88) Months Months Months Months E Lung cancer F Lymphoma GHPancreatic cancer Renal clear cell carcinoma (GSE30219) (GSE10846) (TCGA) (TCGA) MZF1 MZF1 MZF1 MZF1 P=1.2×10-2 P=1.2×10-5 P=6.4×10-4 P=1.5×10-11

Low (n=52) Low (n=189) Low (n=53) Low (n=327)

High (n=362) High (n=88) Percent survival Percent survival Percent survival High (n=75) High (n=123) Percent survival

Months Months Months Months HK2 HK2 HK2 HK2 P=1.3×10-4 P=3.4×10-7 P=6.0×10-3 P=3.0×10-2

Low (n=57) Low (n=216) Low (n=48) Low (n=333)

High (n=198) High (n=82) Percent survival Percent Percent survival Percent Percent survival Percent High (n=207) High (n=128) survival Percent

Months Months Months Months PGK1 PGK1 PGK1 PGK1 P=3.7×10-5 P=2.9×10-2 P=5.7×10-3 P=1.7×10-4 Low (n=297) Low (n=130) Low (n=127) Low (n=363) High (n=117) Percent survival survival Percent survival Percent

High (n=134) High (n=49) survival Percent High (n=52) Months Months Months Months

Figure S3. Kaplan–Meier survival plots of MZF1, HK2, and PGK1 in public tumor datasets. Kaplan–Meier survival plots of patients with low or high levels of MZF1, HK2, and PGK1 in TCGA or GEO datasets of breast cancer (A), endometrial carcinoma (B), glioma (C), head and neck carcinoma (D), lung cancer (E), lymphoma (F), pancreatic cancer (G), or renal clear cell carcinoma (H). Log-rank test for survival comparison.

A Glycolytic function SK-N-AS SK-N-AS 250 dCas9a-CTL dCas9a-MZF1 #1 200 dCas9a-MZF1 #2 Oligomycin ** 150 2-DG Glucose ** ** 100 50 ECAR ECAR (mPH/min) ECAR (mPH/min) ECAR ECAR (mPH/min) ECAR ECAR (mPH/min) ECAR 0 0 20406080100 Time (min) Glycolytic function IMR-32 IMR-32 200 sh-Scb sh-MZF1 #1 160 sh-MZF1 #2 Oligomycin 120 2-DG Glucose 80 ** ** 40 ** ** ECAR (mPH/min) ECAR 0 (mPH/min) ECAR (mPH/min) ECAR 0 20406080100 Time (min)

B SH-SY5Y SK-N-AS

** ** ** ** OCR (pmol/min) OCR (pmol/min) ** **

BE(2)-C IMR-32

160 sh-Scb sh-MZF1 #1 120 sh-MZF1 #2 ** ** ** ** ** 80 * * 40 OCR (pmol/min) 0 (pmol/min)OCR C D SK-N-AS IMR-32 8 Mock MZF1 6 ** 4 ** ** uptake uptake uptake uptake 2 ** ** ** Relative glucose Relative glucose Relative glucose Relative glucose ** 0

** ** ** production production production production ** ** Relative lactate Relative lactate lactate Relative lactate Relative Relative lactate lactate Relative ** **

** ** ** levels levels levels levels ** ** ** ** Relative cellular ATP ATP cellular Relative Relative cellular ATP ATP cellular Relative ATP cellular Relative ATP cellular Relative

Figure S4. MZF1 promotes aerobic glycolysis of NB cells. (A) Seahorse tracing curves (left panel) and ECAR bars (right panel) of SK-N-AS and IMR-32 cells stably transfected with empty vector (mock), MZF1, scramble shRNA (sh-Scb), sh-MZF1, dCas9a control vector (dCas9a-CTL), dCas9a-MZF1, dCas9i control vector (dCas9i-CTL), or dCas9i-MZF1, and those treated with glucose (10 mmol·L-1), oligomycin (2 μmol·L-1), or 2-deoxyglucose (2-DG, 100 mmol·L-1) at indicated (4 replicates for each point). (B) Seahorse extracellular flux assay indicating the oxygen consumption rate (OCR) in SK-N-AS and IMR-32 cells stably transfected with mock, MZF1, sh-Scb, sh-MZF1, dCas9a-CTL, dCas9a-MZF1, dCas9i-CTL, or dCas9i-MZF1 (n=4). (C and D) Glucose uptake, lactate production, and ATP levels in NB cells stably transfected with mock, MZF1, sh-Scb, sh-MZF1, dCas9a-CTL, dCas9a-MZF1, dCas9i-CTL, or dCas9i-MZF1 (n=4). Student’s t test and ANOVA compared the difference in A-D. * P<0.05, ** P<0.01 vs. mock, sh-Scb, dCas9a-CTL, or dCas9i-CTL. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in A-D.

1 1 2 A 1 # 1 # # #2 1 1 2 # 2 2 # K K cb K K K G k -S -H -H H -HK2 #2 P -PG c h-Scb h- h K2 GK1 K2 sh sh sh sh-PGK1sh-PGK1 s #2 sh- sh s s Mo H P Mock H PGK1 - - - --+ + +++ MZF1 ---+ + + sh-MZF1 #1 53 MZF1 53 MZF1

102 HK2 102 HK2

45 PGK1 45 PGK1 42 -actin 42 -actin SH-SY5Y BE(2)-C

B SH-SY5Y BE(2)-C

* * *

uptake uptake Relative glucose * *** * glucose Relative ** ______+ + ++++MZF1 +++ sh-MZF1 #1 1 k cb #1 #2 2 1 #2 #1 Scb DG oc K -S 2 2 1 -DG - 2 # 2 HK GK1 HK2 K 2 K K 2- Mock P M PG sh H sh -H H h- h-HK s s sh sh- sh-PGKsh-PGK1 #2 sh-PGK1sh-PGK1 #1 #2

* * *

production production Relative lactate lactate Relative * * * * * lactate Relative ** ______+ + ++++MZF1 +++ sh-MZF1 #1 G 1 G 1 #1 #2 # #2 ck -Scb 2 1 -D -Scb 2 2 o HK2 2 2-D Mock HK2 M GK1 sh sh PGK P -HK -HK GK1 GK1#1 #2 h-HK2 #2 h P P sh-HKs h-PGK sh s sh-PGK1s #1 sh- sh-

* * *

levels levels * * * ** ** Relative cellular ATP cellular Relative ATP ______ATP cellular Relative _ __ + + ++++MZF1 +++ sh-MZF1 #1 1 2 1 2 k 1 # # #1 #2 cb # # 1 S oc K K -Scb 1 -DG - HK2 HK2 G K K1 2 2-DG M PG Mock P sh HK2 HK2 G G sh -HK2 HK2 h- h- -P -P s s h h sh sh- s s sh-PGK1sh-PGK1 #1 #2

Figure S5. MZF1 promotes aerobic glycolysis of NB cells via up-regulating HK2 and PGK1. (A) Western blot assay indicating the expression of MZF1, HK2, and PGK1 in SH-SY5Y and BE(2)-C cells stably transfected with empty vector (mock), MZF1, scramble shRNA (sh-Scb), or sh-MZF1, and those co-transfected with sh-HK2, sh-PGK1, HK2, or PGK1. (B) Glucose uptake, lactate production, and ATP levels in SH-SY5Y and BE(2)-C cells stably transfected with mock, MZF1, sh-Scb, or sh-MZF1, and those co-transfected with sh-HK2, sh-PGK1, HK2, and PGK1, or treated with 2-DG (10 mmol·L-1) for 48 hrs. ANOVA compared the difference in B. * P<0.05, ** P<0.01 vs. mock+sh-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in A and B.

A sh-HK2 sh-PGK1 sh-Scb #1 #2 #1 #2 2-DG Mock HK2 PGK1

Mock sh-Scb

MZF1 sh-MZF1 #1 SH-SY5Y BE(2)-C

* * *

per per well per well per * * * * * * Number of colonies ______of colonies Number _ __ + + ++++MZF1 +++ sh-MZF1 #1 2 1 2 1 1 2 G 2 2 1 # cb #2 # ck K K -Scb 2 -DG S 2 1 -D o ock H 2 2 M HK GK1 M G sh sh- P P -HK2- HK#1 GK1GK1 # # HK2- HK# GK1 GK# -P -P -P -P sh sh sh- sh sh sh sh sh

B sh-HK2 sh-PGK1 #2 PGK1 sh-Scb #1 #1 #2 2-DG Mock HK2

Mock sh-Scb

MZF1 sh-MZF1 #1 SH-SY5Y BE(2)-C

* * * per per field per field * * * ** * Relative invaded cells Relative Relative invaded cells Relative ______+ + ++++MZF1 +++ sh-MZF1 #1 1 1 G 1 k k #2 # #2 c K2 c 1 2 1 H HK2 K2 # 2-D K 2-DG Mo PGK1 Mo PGK1 sh-ScbH HK2 #2 GK sh-Scb-H sh- sh- sh sh-HK2 #2 sh-PGK1sh-P # sh-PGK1sh-PGK #1

Figure S6. MZF1 promotes growth and invasion of NB cells via up-regulating HK2 and PGK1. Representative images (upper panel) and quantification (lower panel) of soft agar (A) and matrigel invasion (B) assays showing the growth and invasion of SH-SY5Y and BE(2)-C cells stably transfected with empty vector (mock), MZF1, scramble shRNA (sh-Scb), or sh-MZF1, and those co-transfected with sh-HK2, sh-PGK1, HK2, and PGK1, or treated with 2-DG (10 mmol·L-1) for 48 hrs. ANOVA compared the difference in A and B. * P<0.05 vs. mock+sh-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in A and B.

A B

Mock+PBS sh-Scb MZF1+PBS sh-MZF1 #1 * sh-MZF1 #2 Mock+2-DG MZF1+2-DG * * * Tumor weight (g) Tumor Tumorweight (g)

) ) 3 2.5 Mock+PBS 10 Mock+PBS 3 MZF1+PBS MZF1+PBS 2.0 Mock+2-DG 8 Mock+2-DG MZF1+2-DG MZF1+2-DG 1.5 6 ** * * * * 1.0 4 * * ** 0.5 ** 2 ** ** ** levels (qRT-PCR) levels levels (qRT-PCR) levels Relative transcript 0.0 0 Relative transcript Tumor (cm volume Tumor Tumor (cmvolume Tumor 01234 1 1 (week) ZF HK2 (week) ZF1 HK2 M PGK M PGK1

Mock MZF1 sh-Scb sh-MZF1 #1 sh-MZF1 #2

PBS * Ki-67 * * * cells(percentage) cells(percentage) 2-DG Positive expression Ki-67 Positive expression SH-SY5Y BE(2)-C Ki-67

C D Mock_ MZF1_ Mock MZF1 sh-MZF1 ++2-DG sh-Scb #1 #2

100 * * * * * * 80 60 40 sh-Scb 20 sh-MZF1 #1

Percent survival * Metastatic count Metastatic count sh-MZF1 #2 * Metastatic count Metastatic count 0 0 1020304050 Days

Figure S7. MZF1 promotes NB progression via increasing aerobic glycolysis in vivo. (A and B) Representative images, in vivo growth curve, weight at the end points, transcript levels of MZF1, HK2 or PGK1, images and quantification of Ki-67 immunostaining within subcutaneous xenograft tumors formed by SH-SY5Y (A) or BE(2)-C (B) cells stably transfected with empty vector (mock), MZF1, scramble shRNA (sh-Scb), or sh-MZF1 in nude mice, and those treated with daily oral gavage of 2-DG (1 g·kg-1, n=5 for each group). (C and D) Representative images (upper panel) and quantification (left lower panel) of lung metastatic colonization and Kaplan-Meier curves (right lower panel) of nude mice treated with tail vein injection of SH-SY5Y (C) or BE(2)-C (D) cells stably transfected with mock, MZF1, sh-Scb, or sh-MZF1, and those treated with daily oral gavage of 2-DG (1 g·kg-1, n=5 for each group). ANOVA compared the difference in A-D. Log-rank test for survival comparison in C and D. * P<0.05, ** P<0.01 vs. mock+PBS or sh-Scb. Data are shown as mean ± s.e.m. (error bars) in A-D.

P tide F A B C ep -G SH-SY5Y SH-SY5Y BE(2)-C p cb ORF S uPEP GFP u 10 10 uORF 10 uORF 130 kDa MZF1 MZF1 8 8 8 70 kDa 55 kDa 6 * * * 6 * * 6 40 kDa * 35 kDa 4 4 4 25 kDa

(qRT-PCR) 15 kDa 2 2 2 levels (qRT-PCR) levels 10 kDa * * * Relative MZF1 transcript MZF1 Relative transcript 0 transcriptRelative levels 0 0 1 1 F F #2 Z Scb Mock CDS - 1 MZ -MZF1-M RF Mock uORF sh ORF #1 Del u uO MZF UTR- Mut - '- 5 RF sh- sh- ORF- u uO D

30 - IGF1 + IGF1 * 20

* * * 10 * * * * 0 MZF1-AS1 of Percentage 12345678910111213141516 Top Fraction Bottom

E 1 F BS _PBS _IGF P IGF1 + + LY294002 t P ++ ++N-3Flag-uORF pu G In Ig uPE 56 kDa p-AKT IP: _ _ _ WB: + + + IGF1 56 kDa AKT 65 kDa YY1 5 kDa uPEP 37 kDa USF2 82 kDa MZF1 BE(2)-C

42 kDa -actin BE(2)-C

G H TSS YY1

AS C 2 Y5Y 3 MZF1 (−1530/+79) Luciferase K293 -S (2)- H S IP: uPEP MCF 10AHE DG SK-N-BE IMR- WT (-74/-47) 5'-TCCCCAGGTTACCATGGAGACGTTCAGG-3' WB: 65 kDa YY1 Mut (-74/-47) 5'-TCCCCAGGTTATTGCGGAGACGTTCAGG-3' Input: 65 kDa YY1 42 kDa -actin

ratio) ratio) * 1 #2 * Renilla k cb Renilla c 1 S -YY1 h Mo YY sh- sh-YY1 #s

65 kDa (Firefly/ YY1 (Firefly/ * * _ _ _ _ Relative luciferase activity activity luciferase Relative 42 kDa -actin activity luciferase Relative ++_ _ WT ++_ _ Mock ++Mut ++uORF BE(2)-C SH-SY5Y 1 Y1 ck ck Y o YY o Mock M YY1 Mock M YY1 Figure S8. Roles of MZF1, MZF1-uORF, and YY1 in NB. (A) Real-time qRT-PCR assay showing the levels of MZF1 (normalized to -actin, n=4) in SH-SY5Y cells transfected with empty vector (mock), MZF1 coding sequence (CDS), MZF1 containing wild-type, mutant, or deletion forms of 5'-UTR, scramble shRNA (sh-Scb), sh-uORF, or MZF1-uORF. (B) Real-time qRT-PCR assay revealing the levels of uORF and MZF1 (normalized to -actin, n=4) in SH-SY5Y and BE(2)-C cells stably transfected with sh-Scb, sh-uORF, mock, or uORF. (C) Coomassie blue staining showing the expression in BE(2)-C cells transfected with GFP or MZF1-uORF-GFP, with synthesized scramble (Scb) peptide or uPEP as controls. (D) Sucrose gradient sedimentation assay indicating the distribution of uORF or MZF1-AS1 transcript to polysome fractions in SH-SY5Y cells without or with IGF1 (10 nmol·L-1) stimulation for 48 hrs. (E) Western blot assay showing the levels of p-AKT, uPEP, and MZF1 in BE(2)-C cells stably transfected with 3Flag-tagged MZF1-uORF and treated with IGF1 (10 nmol·L-1) or LY294002 (10 μmol·L-1) for 48 hrs. (F) Co-IP and western blot assays revealing the interaction of MZF1-uPEP with YY1 or USF2 in BE(2)-C cells with or without IGF1 (10 nmol·L-1) treatment for 48 hrs. (G) Co-IP and western blot assays indicating the interaction of uPEP with YY1 in non-transformed MCF 10A cells, transformed normal HEK293 cells, normal dorsal root ganglia (DG), and NB cells, and YY1 levels in BE(2)-C and SH-SY5Y cells stably transfected with mock, YY1, sh-Scb, or sh-YY. (H) Dual-luciferase assay showing the activity of MZF1 promoter reporter with wild-type (WT) or mutant (Mut) MZF1-binding site in BE(2)-C cells transfected with mock or YY1, and those stably transfected with MZF1-uORF (n=5). ANOVA and Student’s t test compared the difference in A, B, D and H. * P<0.05 vs. mock, sh-Scb, -IGF1, or mock+WT. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in A-H.

A

** ** ** levels uptake

production Relative lactate Relative glucose * * * __ __ ATP cellular Relative __ ++IGF1 ++IGF1 ++IGF1 BE(2)-C BE(2)-C BE(2)-C

B Mock uORF Mock uORF

PBS PBS ** ** per well per per field IGF1 IGF1 * * Number of colonies

_ _ Relative cells invaded __ + + IGF1 + + IGF1 BE(2)-C BE(2)-C

C D Mock uORF Mock uORF Mock+PBS _ _ ++IGF1 uORF+PBS * Mock+IGF1

uORF+IGF1 *

Tumor weight (g) Tumor * *

) 3 8 Mock+PBS uORF+PBS 6 Mock+IGF-1 * uORF+IGF-1 Metastatic count * 4 * * ** 2 levels (qRT-PCR)

Relative transcript * * *

Tumor (cmTumor volume 0 1 2 1 (week) ZF HK M PGK

Mock uORF * *

PBS * * Percent survival cells (percentage) Positive expression Positive IGF1 BE(2)-C Ki-67

Figure S9. MZF1-uPEP inhibits growth and aggressiveness of NB cells via repressing aerobic glycolysis. (A) Glucose uptake, lactate production, and ATP levels in BE(2)-C (B) cells stably transfected with empty vector (mock) or MZF1 uORF, and those treated with IGF1 (10 nmol·L-1) for 48 hrs. (B) Representative images (left panel) and quantification (right panel) of soft agar and matrigel invasion assays showing the growth and invasion of BE(2)-C cells stably transfected with mock or MZF1 uORF, and those treated with IGF1 (10 nmol·L-1) for 48 hrs. (C) Representative images, in vivo growth curve, weight at the end points, transcript levels of MZF1, HK2 or PGK1, images and quantification of Ki-67 immunostaining within subcutaneous xenograft tumors formed by BE(2)-C cells stably transfected with mock or MZF1 uORF in nude mice, and those treated with daily subcutaneous injection of IGF1 (5 mg·kg-1, n=5 for each group). (D) Representative images (upper panel) and quantification (middle panel) of lung metastatic colonization and Kaplan-Meier curves (lower panel) of nude mice treated with tail vein injection of BE(2)-C cells stably transfected with mock or MZF1 uORF, and those treated with daily subcutaneous injection of IGF1 (5 mg·kg-1, n=5 for each group). ANOVA compared the difference in A-D. * P<0.05, ** P<0.01 vs. mock+PBS. Data are shown as mean ± s.e.m. (error bars) in A-D.

AB uORF --++ BE(2)-C YY1 --+ + 65 kDa YY1

82 kDa MZF1 * * * * *

102 kDa HK2 (qRT-PCR) * 45 kDa PGK1 * * * Relative transcript levels 42 kDa -actin Y1 K2 Y H MZF1 PGK1 BE(2)-C

sh-uORF #1 --++ sh-YY1 #1 --+ + SH-SY5Y 65 kDa YY1

82 kDa MZF1

102 kDa HK2 * * *

(qRT-PCR) 45 kDa PGK1

42 kDa -actin * * * * * Relative transcript levels 1 2 K K1 SH-SY5Y YY1 H MZF PG CE BE(2)-C SH-SY5Y Mock uORF

Mock * * * uptake per well per

Relative glucose * *

* YY1 Numberof colonies BE(2)-C BE(2)-C SH-SY5Y sh-Scb sh-uORF #1

* * sh-Scb

production * per well per Relative lactate * * * BE(2)-C SH-SY5Y Number of colonies #1sh-YY1 SH-SY5Y F Mock uORF *

levels * Mock * * * Relative cellular ATP

per well

YY1 * D Number of colonies BE(2)-C SH-SY5Y BE(2)-C 10 Mock+Mock sh-Scb sh-uORF #1 uORF+Mock 8 Mock+YY1 uORF+YY1 6

** ** 4 sh-Scb

** * ** 2 per well Relative cell viability 0 * (hr) (hr) Numberof colonies 0 24487296 #1sh-YY1 SH-SY5Y

Figure S10. MZF1-uPEP exerts tumor suppressive roles by repressing YY1. (A and B) Western blot (A) and real-time qRT-PCR (B, normalized to -actin, n=4) assays revealing the protein and transcript levels of YY1, MZF1, HK2, and PGK1 in NB cells stably transfected with empty vector (mock), MZF1-uORF, scramble shRNA (sh-Scb), or sh-uORF #1, and those co-transfected with YY1 or sh-YY1 #1. (C) Glucose uptake, lactate production, and ATP levels in NB cells stably transfected with mock, MZF1-uORF, sh-Scb, or sh-uORF #1, and those co-transfected with YY1 or sh-YY1 #1 (n=4). (D) MTT colorimetric assay showing the viability of BE(2)-C and SH-SY5Y cells stably transfected with mock, MZF1-uORF, sh-Scb, or sh-uORF #1, and those co-transfected with YY1 or sh-YY1 #1 (n=6). (E and F) Representative images (left panel) and quantification (right panel) of soft agar (E) and matrigel invasion (F) assays indicating the anchorage-independent growth and invasion capability of NB cells stably transfected with mock, MZF1-uORF, sh-Scb, or sh-uORF #1, and those co-transfected with YY1 or sh-YY1 #1 (n=4). ANOVA compared the difference in B-F. * P<0.05, ** P<0.01 vs. mock or sh-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in A-F.

-4 A Days post-tumor transplantation 3.0×10

0 7 111418 21 28 LV-Mock 2.1×10-4

Tumor cell Virus Sacrifice 1.2×10-4 injection treatment LV-uORF

3.0×10-3 IMR-32 )

3 Ki-67 2.0 LV-Mock LV-uORF 1.5 1.0 LV-Mock

** 0.5 ***

(g) weight Tumor 0.0 *** (cm volume Tumor 01234 Cells (percentage) Cells

(week) Positive expression LV-uORF

B C LV-Mock LV-uORF uPEP 12345 12345 LV-Mock LV-uORF 82 kDa MZF1

LV-Mock *** 102 kDa HK2

45 kDa PGK1 Cells (percentage) Cells Positive expression

LV-uORF 42 kDa -actin D E

2.0 LV-Mock LV-uORF 1.5 1.0 levels uptake (qRT-PCR) 0.5 production

** ** ** *** Relative lactate *** *** Relative glucose glucose Relative

0.0 ATP cellular Relative

Relative transcript levels 1 1 F K HK2 MZ PG F -4 Days post-tumor transplantation 1.5×10 LV-Mock × -4 0714 21 28 35 45 1.2 10

-3 8.3×10 LV-uORF -3 Tumor cell Virus Sacrifice 5.0×10 injection treatment

LV-Mock LV-uORF

* 100 80

20X 60 40 P=0.0345 20 LV-M ock Metastatic count Percent survival Percent LV-uORF 0 0 10 20 30 40 50 100X Days

Figure S11. Therapeutic over-expression of MZF1-uORF inhibits NB progression. (A) In vivo imaging (upper right panel), growth curve (lower left panel), representative images and weight at the end points (lower middle panel), and immunohistochemical staining of Ki-67 (lower right panel) of IMR-32 cells-formed subcutaneous xenograft tumors in athymic nude mice (n=5 per group) that received tail vein injection of lentivirus (LV)-mediated empty vector (mock) or MZF1-uORF as indicated (upper left panel). Scale bar: 100 μm. (B-D) Immunohistochemical staining (B), western blot (C) and real-time qRT-PCR (D, normalized to β-actin) assays indicating the expression of MZF1-uPEP and downstream genes within IMR-32 cells-formed subcutaneous xenograft tumors in nude mice that received tail vein injection of LV-mediated mock or MZF1-uORF (n=5 per group). Scale bar: 100 μm. (E) Glucose uptake, lactate production, and ATP levels of IMR-32 cells-formed subcutaneous xenograft tumors in nude mice (n=5 per group) that received intravenous injection of LV-mediated mock or MZF1-uORF. (F) LV-mediated therapy timeline (upper left panel), in vivo imaging of lungs (upper right panel), representative images and metastatic counts of lungs (lower left and middle panels), and Kaplan-Meier curves (lower right panel) of nude mice (n=5 per group) treated with tail vein injection of IMR-32 cells and LV-mediated mock or MZF1-uORF. Scale bar: 100 μm. Student’s t test and ANOVA compared the difference in A, B and D-F. Log-rank test for survival comparison in F. * P<0.05, ** P<0.01, *** P<0.001 vs. LV-Mock. Data are shown as mean ± s.e.m. (error bars).

AB YY1 Neutralizing peptide Isotype control (GSE62564) P=4.9×10-12 Low (n=268)

High (n=230) MZF1-uPEP

Percent survival

Months

C Breast cancer Endometrial carcinoma Gliomas Head and neck cancer (TCGA) (TCGA) (TCGA) (TCGA) 100 P=3.1×10-3 P=6.1×10-3 × -11 P=1.6 10 P=1.2×10-2 80 Low (n=149) Low (n=298) 60 Low (n=546) 40 Low (n=112) High (n=98) High (n=113) 20 High (n=664) Percent survival Percent survival Percent survival High (n=171) 0 Percent survival 0 100 200 300 Months Months Months Months

Lung cancer Lymphoma Pancreatic cancer Renal clear cell carcinoma (GSE30219) (GSE10846) (TCGA) (TCGA) P=5.5×10-3 P=1.0×10-3 P=1.4×10-4 P=3.0×10-4

Low (n=248) Low (n=195) Low (n=343) Low (n=154) High (n=166) Percent survival High (n=69) Percent survival High (n=22) High (n=72) Percent survival Percent survival

Months Months Months Months

Figure S12. Expression of MZF1-uPEP and YY1 in NB tissues. (A) Representative images of immunohistochemical staining showing the expression of MZF1-uPEP in NB specimens incubated with neutralizing antigen peptide or IgG isotype control. Scale bars: 50 m. (B) Kaplan-Meier curve showing the overall survival of 498 NB patients (GSE62564) with high or low YY1 expression (cutoff value=47.2). (C) Kaplan–Meier survival plots of patients with low or high levels of YY1 in TCGA or GEO datasets of breast cancer, endometrial carcinoma, glioma, head and neck carcinoma, lung cancer, lymphoma, pancreatic cancer, or renal clear cell carcinoma. Log-rank test for survival comparison in B and C.

Table S1 Primer sets used for qRT-PCR and ChIP

Primer set Primers Sequence Product size Application MZF1 Forward 5'-CTGAAACTGAGCCTCCAACTCC-3' 232 qRT-PCR Reverse 5'-CCAGTCTTGCTGTGGGGAAA-3' MZF1-uORF Forward 5'-ATGGAGACGCGGTGGGGCA-3' 63 qRT-PCR Reverse 5'-GCAGGATCCTGCACCAATCA-3' ALDOC Forward 5'-ATGGTGTTCCCTTCGTCCGA-3' 114 qRT-PCR Reverse 5'-GCCCTTGAGTGGTGGTTTCT-3' ENO1 Forward 5'-GATGACTGGGGAGCTTGGCAGAAG-3' 134 qRT-PCR Reverse 5'-TTGAGCAGGAGGCAGTTGCAGGAC-3' GPI Forward 5'-AGTTCTGGGATTGGGTGGGA-3' 230 qRT-PCR Reverse 5'-ATAGGGCAGCATGGCGTGTG-3' HK2 Forward 5'-TGGAGCGAGGTCTGAGCAAG-3' 145 qRT-PCR Reverse 5'-ACCAGCAGGACCCGGAAATT-3' LDHA Forward 5’-TGTCTCTGGCAAAGTGGAT-3 117 qRT-PCR Reverse 5’-ATTAGGTAACGGAATCGGG-3 PGK1 Forward 5'-AGCCAAGATTGTCAAAGACCT-3' 255 qRT-PCR Reverse 5'-GCTTCCCATTCAAATACCCC-3' MZF1-AS1 Forward 5'-GGAATCTGCGTTTATGAAGGGGA-3' 197 qRT-PCR Reverse 5'-TTCTGGGAAGTGTATTTCGGGAC-3' β-actin Forward 5'-TGCCCATCTACGAGGGGTATG-3' 156 qRT-PCR Reverse 5'-TCTCCTTAATGTCACGCACGATTT-3' HK2 Forward 5’-AATACCCCAGCATTTACCAATA-3’ 178 ChIP (-467/-290) Reverse 5’-CTGCCCTTGTCTTACTCCTTCT-3’ PGK1 Forward 5’-ATTGATAACAGCCCTTTCCCCTTCT-3’ 162 ChIP (-1507/-1346) Reverse 5'-ATGGTGATGTTATTCCCAGAAGCAA-3' MZF1 Forward 5’-CCCCACCTCTTCATCCCACAT-3’ 188 ChIP (-156/+32) Reverse 5’-GTAGTCAGCAGGCCCTTCGTG-3’ MZF1, myeloid zinc finger 1; uORF, upstream open reading frame; ALDOC, aldolase, fructose-bisphosphate C; ENO1, enolase 1; GPI, glucose-6-phosphate isomerase; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; PGK1, phosphoglycerate kinase 1; MZF1-AS1, myeloid zinc finger 1 antisense RNA 1.

Table S2 Oligonucleotide sets used for constructs

Primer set Primers Sequence dCas9a-MZF1 #1 Forward 5’-CACCGGACACGACTCGACATCGCCTGGG-3’ Reverse 5’-AAACCCCAGGCGATGTCGAGTCGTGTCC-3’ dCas9a-MZF1 #2 Forward 5’-CACCGGGGTAACCCGGAGACGCGGCGGG-3’ Reverse 5’-AAACCCCGCCGCGTCTCCGGGTTACCCC-3’ dCas9i-MZF1 #1 Forward 5’-CACCGGGCCGTCATTAGAACTCCGTCGG-3’ Reverse 5’-AAACCCGACGGAGTTCTAATGACGGCCC-3’ dCas9i-MZF1 #2 Forward 5’-CACCGCACCGACGGAGTTCTAATGACGG-3’ Reverse 5’-AAACCCGTCATTAGAACTCCGTCGGTGC-3’ pGL3-5'-UTR Forward 5’-GGAAGATCTGCGCACGTACACACGAAGGGCCTG-3’ Reverse 5’-CCCAAGCTTAGAGGGTACCAGGTCAGGTATCTG-3’ pGL3-P-5'-UTR Forward 5’-GGAAGATCTTCACAGTGCAGTTTTCCGGAGGACG-3’ Reverse 5’-CCCAAGCTTAGAGGGTACCAGGTCAGGTATCTG-3’ pGL3-5'-UTR Mut Forward 5’-GGGAGTTGCCTAGGAGACGCGGTGGGGCACCGACGGAGTTCTA-3’ Reverse 5’-ACCGCGTCTCCTAGGCAACTCCCTACAGGAAGGGGCCTCTGCT-3’ pGL3-5' UTR Del Forward 5’-AGTTGCCATGAGACGCGGTGGGGCACCGACGGAGTTCTAAT-3’ Reverse 5’-CCACCGCGTCTCATGGCAACTCCCTACAGGAAGGGGCCTCT-3’ pcDNA3.1-MZF1 CDS Forward 5’-CCCAAGCTTATGAGGCCTGCGGTGCTGGGCT-3’ Reverse 5’-CCGGAATTCCTACTCGGCGCTGTGGACGCGC-3’ pcDNA3.1-5'-UTR-MZF1 Forward 5’-CCCAAGCTTGCGCACGTACACACGAAGGGCC-3’ Reverse 5’-CCGGAATTCGGGTGGAGACCCAGTTTCCATC-3’ pcDNA3.1-5'-UTR-MZF1 Mut Forward 5’-GGGAGTTGCCTAGGAGACGCGGTGGGGCACCGACGGAGTTCTA-3’ Reverse 5’-ACCGCGTCTCCTAGGCAACTCCCTACAGGAAGGGGCCTCTGCT-3’ pcDNA3.1-5'-UTR-MZF1 Del Forward 5’-AGTTGCCATGAGACGCGGTGGGGCACCGACGGAGTTCTAAT-3’ Reverse 5’-CCACCGCGTCTCATGGCAACTCCCTACAGGAAGGGGCCTCT-3’ pCMV-3Tag-1A-MZF1 uORF Forward 5'-CCCAAGCTTATGGAGACGCGGTGGGGCA-3' Reverse 5’-GCCGCTCGAGTTAGCAGGATCCTGCACCAA-3’ pCMV-C-Tag-MZF1 uORF Forward 5’-CCCAAGCTTATGGAGACGCGGTGGGGCA-3 Reverse 5’-GCCGCTCGAGGCAGGATCCTGCACCAATCA-3 pEGFP-N1-MZF1-uORF Forward 5’-GCCGCTCGAGATGGAGACGCGGTGGGGCA-3 Reverse 5’-CCCAAGCTTGCAGGATCCTGCACCAATCA-3’ pEGFP-N1-MZF1 uORF Mut Forward 5’-AGATCTCGAGTAGGAGACGCGGTGGGGCACCGACGGAGTTCTA-3' Reverse 5’-ACCGCGTCTCCTACTCGAGATCTGAGTCCGGTAGCGCTAGCGG-3’ pEGFP-N1-MZF1 uORF Del Forward 5’-TCTCGAGATGAGACGCGGTGGGGCACCGACGGAGTTCTAAT-3’ Reverse 5’-CCACCGCGTCTCATCTCGAGATCTGAGTCCGGTAGCGCTAG-3’ pEGFP-N1 Mut Forward 5’-ACCGGTCGCCTCCCTGTTGAGCAAGGGCGAGGAGCTGTTCACCGGGGT-3’ Reverse 5’-GCCCTTGCTCAACAGGGAGGCGACCGGTGGATCCCGGGCCCGCGGTAC-3’ pCMV-N-MYC-YY1 Forward 5’-CCGGAATTCATGGCCTCGGGCGACACCCTCTA-3’ Reverse 5’-GCCGCTCGAGTCACTGGTTGTTTTTGGCCTTAG-3’ pCMV-N-MYC-YY1 Forward 5’-CCGGAATTCATGGCCTCGGGCGACACCCTCTA-3’ (N-terminal) Reverse 5’-GCCGCTCGAGCGGGTCGGCGCCGCCGCCGCCCG-3’ pCMV-N-MYC-YY1 Forward 5’-CCGGAATTCATGGCCTCGGGCGACACCCTCTA-3’ (ΔZFN) Reverse 5’-GCCGCTCGAGTGTCATATATTCTGAATAATCAG-3’ pCMV-N-MYC-YY1 Forward 5’-CCGGAATTCGGCAACAAGAAGTGGGAGCAGAA-3’ (Δ-N-terminal) Reverse 5’-GCCGCTCGAGTCACTGGTTGTTTTTGGCCTTAG-3’ pCMV-N-MYC-YY1 Forward 5’-CCGGAATTCGAAAAAAAAGATATTGACCATGA-3’ (C-terminal) Reverse 5’-GCCGCTCGAGTCACTGGTTGTTTTTGGCCTTAG-3’ pLV-MZF1 uORF Forward 5’-TGCTCTAGAATGGATTACAAGGATGACGACG-3’ Reverse 5’-ATTTGCGGCCGCTTAGCAGGATCCTGCACCAATC-3’ pGL3-YY1 Forward 5’-CCGGCCATCTTGATACGACCATCTTCTATCGGCCATCTTGATACGACCATGTTCA-3’ Reverse 5’-AGCTTGAACATGGTCGTATCAAGATGGCCGATAGAAGATGGTCGTATCAAGATGGCCGGGTAC-3’ pGL3-MZF1 (-1530/+30) Forward 5’-CGGGGTACCTCCCGGGTTCACGCCATTCTCCTGC-3’

Reverse 5’-GCCGCTCGAGTAGACCGCAAAATGGAGAAAGGCTT-3’ pGL3-MZF1 Mut Forward 5’-CCCCAGGTTATTGCGGAGACGTTCAGGGCCCGCCCCTCGCCCATG-3’ Reverse 5’-TGAACGTCTCCGCAATAACCTGGGGACACGACTCGACATCGCCTG-3’ pGL3-HK2 (-1813/+424) Forward 5’-CAAGGAAAGCCTGATGAGGTAGAAG-3’ Reverse 5’-TGATGATGTGAATGAACTGGGTAGA-3’ pGL3-HK2 Mut Forward 5’-TCAGGAGCCTAGATAGTGAGGTTAGCCAGAAACCCTCAGAGTGGA-3’ Reverse 5’-CTAACCTCACTATCTAGGCTCCTGAGTCCCTGTTTGCTCTATTGG-3’ pGL3-PGK1 (-882/+246) Forward 5’-CGGGGTACCCTAAGAACTTGGACACCCTCCACG-3’ Reverse 5’-GCCGCTCGAGCAGAATTACCTCATAACGACCCGC-3’ pGL3-PGK1 (Mut) Forward 5’-CGACCTCTCTACTGAGCTGTATTTCCAAAATGTCGCTTTCTAACA-3’ Reverse 5’-GAAATACAGCTCAGTAGAGAGGTCGGTGATTCGGTCAACGAGGGA-3’ MZF1, myeloid zinc finger 1; uORF, upstream open reading frame; YY1, Yin Yang 1; HK2, hexokinase 2; PGK1, phosphoglycerate kinase 1.

Table S3 Oligonucleotides encoding short hairpin RNAs

Oligo Set Sequences

sh-Scb 5'-CCGGGCGAACGATCGAGTAAACGGACTCGAGTCCGTTTACTCGATCGTTCGCTTTTT-3' (sense);

5'-AATTCAAAAAGCGAACGATCGAGTAAACGGACTCGAGTCCGTTTACTCGATCGTTCGC-3' (antisense) sh-MZF1 #1 5'-CCGGCCGGCCTGCCTTTGACCCTATCTCGAGATAGGGTCAAAGGCAGGCCGGTTTTTG-3' (sense);

5'-GATCCAAAAACCGGCCTGCCTTTGACCCTATCTCGAGATAGGGTCAAAGGCAGGCCGG-3' (antisense) sh-MZF1 #2 5'-CCGGGACTGGGAGAGAATGCCCAGTCTCGAGACTGGGCATTCTCTCCCAGTCTTTTTG-3' (sense);

5'-GATCCAAAAAGACTGGGAGAGAATGCCCAGTCTCGAGACTGGGCATTCTCTCCCAGTC-3' (antisense) sh-HK2 #1 5'-CCGGTGGAGCTCAACCATGACCAAGTCTCGAGACTTGGTCATGGTTGAGCTCCTTTTTG-3' (sense);

5'-GATCCAAAAAGGAGCTCAACCATGACCAAGTCTCGAGACTTGGTCATGGTTGAGCTCCA-3' (antisense) sh-HK2 #2 5'-CCGGTGGGTGAAAGTAACGGACAATGCTCGAGCATTGTCCGTTACTTTCACCCTTTTTG-3' (sense);

5'-GATCCAAAAAGGGTGAAAGTAACGGACAATGCTCGAGCATTGTCCGTTACTTTCACCCA-3' (antisense) sh-PGK1 #1 5'-CCGGTGCTGTCCCAAGCATCAAATTCCTCGAGGAATTTGATGCTTGGGACAGCTTTTTG-3' (sense);

5'-GATCCAAAAAGCTGTCCCAAGCATCAAATTCCTCGAGGAATTTGATGCTTGGGACAGCA-3' (antisense) sh-PGK1 #2 5'-CCGGTGCCTGACAAGTACTCCTTAGACTCGAGTCTAAGGAGTACTTGTCAGGCTTTTTG-3' (sense);

5'-GATCCAAAAAGCCTGACAAGTACTCCTTAGACTCGAGTCTAAGGAGTACTTGTCAGGCA-3' (antisense) sh-MZF1-uORF #1 5'-CCGGGCAGCTTCATAACCGAAGATTCTCGAGAATCTTCGGTTATGAAGCTGCTTTTTG-3' (sense);

5'-GATCCAAAAAGCAGCTTCATAACCGAAGATTCTCGAGAATCTTCGGTTATGAAGCTGC-3' (antisense) sh-MZF1-uORF #2 5'-CCGGCCGAGAAATCTCTTACCTCAACTCGAGTTGAGGTAAGAGATTTCTCGGTTTTTG-3' (sense);

5'-GATCCAAAAACCGAGAAATCTCTTACCTCAACTCGAGTTGAGGTAAGAGATTTCTCGG-3' (antisense) sh-YY1 #1 5'-CCGGGCTTCACATATCAGCAGGTTACTCGAGTAACCTGCTGATATGTGAAGCTTTTTG-3' (sense);

5'-GATCCAAAAAGCTTCACATATCAGCAGGTTACTCGAGTAACCTGCTGATATGTGAAGC-3' (antisense) sh-YY1 #2 5'-CCGGACCTCTTCGACTGTGACTTTGCTCGAGCAAAGTCACAGTCGAAGAGGTTTTTTG-3' (sense);

5'-GATCCAAAAAACCTCTTCGACTGTGACTTTGCTCGAGCAAAGTCACAGTCGAAGAGGT-3' (antisense) MZF1, myeloid zinc finger 1; HK2, hexokinase 2; PGK1, phosphoglycerate kinase 1; uORF, upstream open reading frame; YY1, YY1 transcription factor.

Table S4 Glycolytic genes and transcription factors differentially expressed in NB (GSE16476)

Differentially expressed glycolytic genes Differentially expressed transcription factors Age Death 2 vs. 4 stage Overlap Age Death 2 vs. 4 stage Overlap ALDOA ALDOA ALDOC ALDOC ATF2 AATF ZNF138 CBFB CBFB ALDOC ALDOC ENO1 ENO1 BUD31 AFF4 ZNF219 E2F3 E2F3 ENO1 ENO1 GPI GPI CBFA2T2 ATF2 ZNF256 E2F8 HMGA1 GPI GPI HK2 HK2 CBFB CBFA2T2 ZNF287 GATAD2A MZF1 HK2 HK2 LDHA LDHA CREB1 CBFB ZNF367 HMGA1 TRIM28 LDHA LDHA PGK1 PGK1 E2F3 CREB1 ZNF496 MYCN ZEB2 PFKM PGK1 PKM PKM ENO1 CREB5 ZNF70 MZF1 ZHX2 PGK1 PKM FOXP1 CREBL2 ZNF83 NR3C1 ZNF496 PKM SLC2A1 HMGA1 DLX5 ZNF93 PLAGL2 ZNF93 HOXC9 DMRT3 ZRANB2 SCML2 KLF5 E2F1 TRIM28 KLF7 E2F3 UHRF1 MZF1 E2F6 ZEB2 NFYA E2F8 ZHX2 NR3C1 ENO1 ZNF496 PA2G4 ESRRA ZNF93 RORA ETV3 SALL1 ETV4 SCAND1 FEV SOX6 FOXC1 TCF7L2 FOXD3 TRIM28 FOXM1 TSHZ2 FOXO3 TULP4 FOXP1 VAX2 FUBP1 VPS72 GATAD2A ZBTB38 HES6 ZEB2 HIF1A ZFP36L2 HMGA1 ZHX2 HMGB2 ZNF138 HMX1 ZNF219 HOXC9 ZNF256 HOXD10 ZNF496 HOXD4 ZNF93 HOXD9 IRX3 IRX5 JUN JUND KLF12 KLF5 KLF7 MTA1 MYBL2 MYT1L MZF1 NFATC3 NFYA NR2C1 NR2F1 PA2G4 PLAGL2 POU4F1 POU6F1 PTTG1 RELA RFXANK RNF4 RORA RORB SCML2 SIX2 SMAD3 SMARCA4 SNAPC2 SOX4 SOX6 SRF TBX1 TCF3 TEAD4 THRA TLX3 TP53 TRIM28 TSHZ2 TULP4 UHRF1 VAX2 YY1 ZBTB38 ZEB2 ZHX2

Table S5 Mass spectrometry analysis of pulled down by MZF1-uPEP

A1BG CACYBP DHDDS GNPDA2 LPL NUP160 PTP4A1 SMAD1 TUBB2B ABCA1 CADM4 DHFR GNPNAT1 LRRC40 NUP188 PTP4A2 SMAD2 TUBG1 ABCC5 CALCOCO2 DHODH GNS LRRC47 NUP43 PTPN1 SMAD3 TUBG2 ABCF3 CAMK1 DHPS GOLGB1 LRRC57 NUP50 PTPRA SMAD4 TUBGCP4 ABHD18 CAMK2A DHRS1 GOLIM4 LRRN2 NUP54 PTPRK SMAD5 TUFM ACACA CAPN1 DHX29 GOLPH3 LSM14B NUP62 PTRF SMAD9 TWF2 ACAD9 CAPZA1 DHX36 GPAT2 LSM2 NUP85 PUM1 SMARCAD1 TXNDC12 ACADS CARF DHX38 GPD1 LSM3 NUTM2F PUS10 SMARCE1 U2AF1 ACBD3 CARM1 DHX57 GPR135 LSM4 NUTM2G PUS7 SMC3 U2AF1L5 ACOT9 CARNMT1 DIAPH3 GRHPR LTA4H NXF2 PWP2 SMC5 U2AF2 ACP1 CASP8AP2 DIMT1 GRIN3B LUC7L3 NXF2B PYGB SMPD4 UAP1 ACSL1 CAT DIP2A GRIPAP1 LUZP1 ODR4 QDPR SMS UBA6 ACTA1 CBX5 DIS3 GRPEL1 LYAR OGT QKI SMYD5 UBE2C ACTA2 CC2D2A DISC1 GRWD1 LZTR1 OR1B1 RAB11FIP2 SNAP23 UBE2D1 ACTC1 CCAR1 DKFZp566H1924 GSTM1 LZTS3 OR2I1P RAB12 SNAPIN UBE2D4 ACTG2 CCAR2 DKFZp686E0752 GSTM3 MAATS1 OSBP RAB14 SNCG UBE2E1 ACTN3 CCBL2 DKFZp686I0230 GSTM4 MAN2A1 OSBPL7 RAB21 SNF8 UBE2E2 ACTR10 CCDC141 DLD GTF2I MAP1LC3B OSBPL8 RAB23 SNRPC UBE2E3 ACTR5 CCDC157 DLGAP4 GTPBP1 MAP1LC3B2 OSBPL9 RAB3A SNRPG UBE2G2 ADAM28 CCDC171 DMXL1 GTSE1 MAP2 OSTF1 RAB3B SNRPGP15 UBE2I ADARB1 CCDC173 DNAH3 GUK1 MAP3K14 OTUD6B RAB3C SNTB2 UBE2K ADH6 CCDC47 DNAH6 GYS2 MAP4K4 OXSR1 RAB3D SNX1 UBE2L3 ADH7 CCDC79 DNAH9 HADH MAP7D3 P2RX4 RAB3GAP2 SNX25 UBE2O ADRA1D CCDC85C DNAJB1 HAT1 MAP9 P3H1 RAB41 SNX6 UBE2T ADRA2B CCDC87 DNAJB11 HCCS MARC2 PACSIN2 RAB44 SNX8 UBE3A ADRM1 CCNE1 DNAJC10 HDAC2 MARS PAFAH1B2 RAB5B SOS2 UBE3C ADSL CDC123 DNAJC19 HEATR1 MAT2B PAIP1 RAB5C SPAG16 UBE4A AGK CDC73 DNAJC21 HECTD1 MCF2L PARP10 RAB6B SPATA31D1 UBE4B AGL CDCA7L DNAJC7 HECTD4 MCTS1 PARVA RAB8A SPATA31D3 UBR3 AGPAT5 CDIPT DNM1L HEL-S-109 MDN1 PBDC1 RAB8B SPATA31D4 UBR5 AGPS CDK2 DNMT1 HELZ ME1 PCBD1 RAD1 SPCS2 UBTF AHR CDK5 DPH7 HGS MEAF6 PCBD2 RAD21 SPIRE1 UBXN2B AIF1L CDK5R2 DPM1 HGSNAT MEPE PCBP3 RAE1 SPIRE2 UBXN4 AIMP2 CDKN2AIPNL DPP10 HID1 MESDC2 PCDHAC2 RAI1 SPNS1 UBXN7 AKAP2 CEBPE DPP9 HIRA METTL22 PCDHB11 RALGAPA2 SPOPL UFC1 AKAP8L CEBPZ DR1 HLA-B MGAT2 PCDHB14 RARS SQSTM1 UFL1 ALDH3A2 CELF2 DRG2 HLA-C MIC13 PCDHGB3 RASA3 SRC UFM1 ALDH3B1 CEP135 DSTN HM13 MIDN PCYT2 RASAL2 SRP14 UGP2 ALDH3B2 CEP162 DTNBP1 HMGB2 MIEN1 PDCD1 RASIP1 SRP54 UGT2B15 ALDH9A1 CEP95 DUPD1 HMGB3 MLEC PDCD11 RASSF5 SRPRA UHMK1 ALF CERS2 DUS3L HMGCL MOB1A PDCL3 RASSF7 SRPRB UHRF1 ALYREF CFAP36 DUSP15 HMGCS1 MOB1B PDE4DIP RAVER1 SRSF10 UHRF1BP1 AMPH CFAP47 DYNC1I2 HMOX2 MOB4 PDK3 RBBP7 SRSF11 UHRF1BP1L ANAPC7 CFAP54 DYNC1LI1 HNRNPA0 MOSPD2 PDLIM2 RBM12 SSB UHRF2 ANGPT4 CFAP58 DYNLT1 HNRNPD MPRIP PDLIM5 RBM22 SSH3 UMODL1 ANKHD1 CHAF1B ECM29 HNRNPH3 MRC2 PDP2 RBM24 SSR1 UNC119 ANKRD17 CHD2 EEF1E1 HNRNPUL1 MRE11A PDS5A RBM25 SSR3 UNC119B ANKRD66 CHD3 EEFSEC HPCA MROH2A PDS5B RBM28 SSR4 UNC13C ANKRD7 CHD5 EFCAB8 HPCAL1 MROH5 PDZD2 RBM38 ST13P5 UNC13D ANKUB1 CHD7 EHBP1 HSFY1 MRPL17 PEAK1 RBM4 ST6GAL2 UNC79 ANO4 CHKB EHD4 HSP90AB3P MRPL19 PELP1 RBPJ STK10 UNKL ANO6 CHMP6 EIF2B2 HSP90B2P MRPL24 PEPD RBX1 STK17A UQCRB ANP32A CHRNB1 EIF3G HSPA14 MRPL32 PEX1 RCC1 STOM USF2 ANP32D CHURC1-FNTB EIF3K HSPA1A MRPL9 PEX14 RDH11 STON1-GTF2A1L USP10 ANXA11 CIAPIN1 EIF3M HSPA1B MRPS16 PFAS RDH13 STRN USP14 ANXA13 CISD1 EIF4A2 HSPE1-MOB4 MRPS18B PFDN5 RDH14 STT3B USP32 ANXA7 CISD2 EIF4G3 IARS2 MRPS22 PFKM REEP4 STX4 USP39 AP1M1 CKS1B EIF4H IDI1 MRPS23 PGD REEP5 STXBP1 USP4 AP2S1 CLASP2 EIF6 IER3IP1 MRPS34 PGK2 RFC5 STXBP4 USP9Y AP3D1 CLDND1 ELAC2 IFIT1 MRTO4 PGLS RHOC STXBP6 UTP4 AP3M1 CLEC16A ELMO2 IFITM1 MSANTD4 PGM2 RIC1 SUPT5H UTRN AP3S1 CLIC2 EMC2 IFITM2 MSTO1 PGS1 RILPL1 SUZ12 UVRAG AP4B1 CLIP1 ENTPD3 IFITM3 MT-CYB PHF2 RILPL2 SYNJ2BP VAPB APOB CLIP2 EP300 IFT122 MTDH PHPT1 RIN1 SYNJ2BP-COX16 VMP1 APP CLN6 EPB41L1 IGF2BP1 MTERF3 PIK3C2B RNPEP TACC2 VPS11

APRT CLNS1A EPHA2 IGHMBP2 MTMR2 PIK3CA ROBO2 TANC2 VPS18 AQR CLPTM1 EPM2AIP1 IGHV3-15 MTMR4 PIN1 RPA1 TANK VPS25 ARF1 CLPX ERC1 IGHV3-72 MTOR PIWIL3 RPA2 TARBP1 VPS26A ARF3 CLTA ERC2 IGHV3-73 MTTP PKD1 RPA3 TAS2R39 VPS26B ARF5 CLTB ERH IL26 MTX1 PKD1L1 RPL27A TATDN1 VPS29 ARF6 CMYA5 ERMP1 IL6ST MTX2 PKN1 RPL36A-HNRNPH2 TAX1BP3 VPS37B ARFGEF2 CNN3 ERVPABLB-1 ILVBL MUC16 PLA2G4A RPL37AP8 TBC1D2 VPS53 ARFIP2 CNOT1 ESPN IMMP2L MUSK PLAA RPL39 TBC1D4 VRK1 ARGLU1 CNOT4 ESPNL IMMT MYCBP2 PLBD2 RPL39P5 TBKBP1 VWA5B2 ARHGAP5 CNPY4 EWSR1 IMPA2 MYDGF PLCB3 RPP25L TBL1X VWA8 ARHGDIB CNTLN EXD3 INA MYH10 PLCD1 RPP30 TBL1Y WAPL ARHGEF19 CNTN5 EXOSC9 INCENP MYH15 PLCG1 RPRD1B TBL3 WDR36 ARHGEF38 CNTNAP3B EYS INTS3 MYH3 PLEKHA6 RPS6KA5 TBRG4 WDR37 ARHGEF7 COG4 FAF2 INTS6 MYL1 PLEKHH1 RPTOR TCEA2 WDR43 ARID3C COG7 FAM104B INTS7 MYL12A PLOD2 RPUSD2 TCHH WDR48 ARIH1 COL18A1 FAM114A1 INTS9 MYL12B PLPPR3 RRAGA TDP1 WDR5 ARIH2 COL1A1 FAM114A2 IPO4 MYNN PLXNB1 RRAGB TEC WDR6 ARL1 COL4A2 FAM129A IPO8 MYO15A PMM1 RRAGC TEP1 WDR60 ARL2 COL4A3BP FAM13A IQCA1 MYO18A PMM2 RRAGD TFEB WDR61 ARL3 COL7A1 FAM161B IQCD MYO5A PMS2P2 RRM2 TFG WDR75 ARL6IP1 COLEC12 FAM162A IQGAP3 MYO6 PNP RRP1 TFR2 WDR77 ARL6IP5 COPB1 FAM170B IREB2 NAA25 PNPLA7 RRP12 TFRC WDR81 ARMC7 COPE FAM188A IRS4 NAA40 POC5 RRP9 TGM1 WDR82 ARPIN COPS7A FAM50A ISCU NAA50 POLA1 RSL24D1 TGM2 WDR87 ARRB1 COPS8 FAM50B ISOC1 NAE1 POLA2 RSPH3 TGM5 WIPI1 AS3MT COX5B FAM98A ISY1 NAGK POLDIP2 RTCA THADA WWP1 ASH1L COX6B1 FAM98B ITCH NAGLU POLR1E RTKN2 THOC3 WWP2 ASL CPNE2 FANCE ITGA5 NANS POLR2G RTN4 TIMM23 XAB2 ASNA1 CPNE3 FBXL2 ITGAD NAP1L1 POLR2H RXFP4 TIMM23B XRN2 ATAD1 CPNE4 FBXL20 ITGAL NAP1L4 POM121C RXRA TJP1 XRRA1 ATE1 CPNE5 FBXL8 ITGB1 NAPA PON3 RXRB TM9SF1 YKT6 ATG16L1 CPNE6 FBXO39 ITIH3 NAPG POTEJ RXRG TMED7 YRDC ATL2 CPNE7 FCAMR IVD NAT10 PPFIA1 S100A10 TMEM11 YTHDC2 ATL3 CPNE8 FCF1 JAK3 NAT16 PPFIBP2 SALL1 TMEM167A YTHDF1 ATP1A3 CPNE9 FCRLB JAKMIP1 NAV2 PPIC SCCPDH TMEM192 YTHDF2 ATP5F1 CPOX FECH KALRN NAV3 PPIG SCFD1 TMEM2 YTHDF3 ATP5I CPSF1 FEN1 KANK4 NCAPD2 PPIL2 SCN8A TMEM5 YY1 ATP5S CPSF2 FERMT2 KAT6B NCDN PPM1D SCP2 TMEM79 YY2 ATP6V0A1 CPSF3 FILIP1L KCMF1 NCK1 PPP1R10 SCYL2 TMLHE ZADH2 ATP6V1C1 CPSF3L FKBP2 KCNAB3 NCOR2 PPP1R12A SDHD TNC ZBED4 ATP6V1F CPT1A FKBP3 KCNK12 NCSTN PPP1R12B SEC22B TNPO2 ZBTB10 ATP9B CPT1C FLVCR1-AS1 KCTD9 NDRG3 PPP1R35 SEC24B TNRC6A ZCCHC2 ATPAF1 CPT2 FN3KRP KIAA0196 NDST1 PPP2R1B SEC24D TNRC6B ZFC3H1 ATR CRELD2 FNBP1 KIAA1033 NDST2 PPP2R2A SEC61A2 TNS2 ZFP42 ATXN2L CRTAP FNIP1 KIAA1549 NDST3 PPP2R2B SEC61B TOE1 ZFP90 ATXN7 CRY1 FNTB KIAA1551 NDST4 PPP2R2D SEC63 TOMM22 ZFP91 ATXN7L2 CRY2 FRMD4B KIAA1671 NDUFA5 PPP4R1 SELENBP1 TOMM34 ZFP91-CNTF BAG2 CRYBB3 FRMPD2 KIF11 NDUFAB1 PPP4R1L SELT TOMM40 ZFR BAG3 CSNK2A2 FRYL KIF13A NDUFB2 PPWD1 SEMA3B TOP3A ZMPSTE24 BAIAP2 CSRP1 FSTL4 KIF15 NDUFB6 PREX2 SEMA6B TOR1B ZNF233 BAX CTNNBL1 FTO KIF1B NDUFB7 PRKAA1 SEP15 TPD52 ZNF462 BCAP31 CTNND2 FTSJ3 KIF1Bbeta NDUFB9 PRKAG1 SEPT12 TPD52L2 ZNF662 BCCIP CTSB FUBP3 KIF21A NDUFS2 PRKAR2A SEPT14 TPGS1 ZNF83 BCL2L10 CTSD FUNDC2 KIF2A NDUFS3 PRKCA SEPT8 TPM1 ZNF853 BECN1 CTSL FUS KIF3A NDUFS7 PRKCB SERBP1 TPP1 ZPR1 BHLHB9 CTSS FUSIP1 KIF3B NDUFS8 PRKCE SERGEF TRA2A ZW10 BICD1 CTSV FXN KIF6 NDUFV1 PRKCH SERINC5 TRA2B BIN2 CTTN GAA KLC1 NEDD8 PRKCQ SERPINA3 TRAP1 BLOC1S4 CTTNBP2 GABBR2 KLHL3 NEDD8-MDP1 PROSC SETD3 TRAPPC11 BLVRB CWF19L1 GALNT10 KMO NEMF PRPF3 SETD7 TRIB3 BNIP1 CYB5A GALNT11 KPNA2 NEXN PRPF31 SF3B2 TRIM24 BPIFB6 CYP2J2 GALNT7 KRR1 NFIB PRPF4 SF3B4 TRIM3 BRD2 CYP4X1 GAS2 KRT10 NFU1 PRPF6 SF3B6 TRIM32 BRD3 DAAM2 GAS8 KRT2 NFYC PRPH SFSWAP TRIM33 BRINP1 DAB1 GCLC LAMB2 NIT2 PRPS1L1 SGK1 TRIM39 BRIP1 DAB2IP GCLM LAMTOR2 NMD3 PRPS2 SH2D4A TRIM39-RPP21 BRIX1 DAD1 GCNT1 LANCL1 NME1 PRPSAP1 SH3GL1 TRIM47

BRK1 DCAF13 GCSH LARP7 NOC2L PRR23A SH3GL2 TRIO BRWD1 DCTN1 GET4 LCLAT1 NOP10 PRR23B SH3GLB1 TRIOBP BYSL DCTN2 GHITM LCMT1 NOXA1 PRR36 SH3GLB2 TRIP11 C11orf95 DCTN3 GLG1 LDHAL6A NPC2 PRRC2A SHISA6 TRIP12 C14orf159 DCTN4 GLRX5 LGALS3BP NPDC1 PRSS1 SIN3A TRMT10A C15orf38-AP3S2 DCUN1D1 GLUL LGALSL NPEPL1 PRSS23 SKIV2L2 TRMT13 C15orf56 DDX18 GLYCTK LIG1 NPTN PSAP SLC12A9 TRMT61A C16orf62 DDX42 GMPPA LILRA6 NRDC PSEN1 SLC15A3 TRNT1 C18orf65 DDX47 GMPPB LIMS1 NRP2 PSEN2 SLC25A11 TRPM2 C19orf70 DDX49 GNA11 LIMS4 NSA2 PSIP1 SLC25A20 TRPM8 C1orf106 DDX54 GNA13 LIN7B NSMCE4A PSMB7 SLC25A27 TSC22D1 C1orf167 DDX6 GNA14 LMTK3 NT5C PSMD7 SLC38A11 TSNAX C21orf2 DEK GNAI1 LNPEP NTMT1 PSMG1 SLC39A12 TTC25 C22orf31 DENND3 GNAI2 LOC100996720 NUCB1 PSMG3 SLC44A1 TTC31 CAB39 DENND4B GNAQ LOC102724159 NUCB2 PSPC1 SLC6A11 TTC34 CACNA1H DERL1 GNAS LONP2 NUCKS1 PTGR2 SLC6A8 TTLL12 CACNA1S DGCR6 GNE LONRF3 NUDCD1 PTGS1 SLK TTLL13P CACNB3 DGCR6L GNL1 LPCAT1 NUDT21 PTMA SLU7 TUBA4A

Table S6 Transcription factors regulating MZF1 revealed by UCSC

ATF1 FOSL2 NFYB STAT3 ATF2 FOXA1 NR3C1 TAF1 ATF3 FOXP2 NRF1 TBP BACH1 GABPA PAX5 TCF12 BHLHE40 IRF1 PBX3 TCF3 CEBPB IRF3 RELA TCF4 CTCF JUN REST TCF7L2 CTCFL JUND RFX5 USF1 E2F1 KDM5B RUNX3 USF2 E2F4 MAX SIX5 YY1 EGR1 MAZ SP1 ZNF143 ELF1 MXI1 SP2 ZNF263 ELK1 MYC SP5 ETS1 NFIC SPI1 FOS NFYA SRF

Table S7 MZF1-uPEP expression in human NB tissues

Group Total number MZF1-uPEP expression Positive rates (%) P-Value − + ++ +++ Age <1 year 20 5 6 5 4 75.0 0.038 ≥1 year 22 15 4 2 1 31.8

Differentiation Well differentiated 8 0 2 3 3 100.0 Poorly differentiated 28 15 7 4 2 46.4 0.024 Undifferentiated 6 5 1 0 0 16.7

MKI <200 17 5 3 5 4 70.6 0.037 >200 25 15 7 2 1 40.0

INSS stages Stage 1-2 14 2 3 5 4 85.7 Stage 3-4 20 15 4 1 0 25.0 0.007 Stage 4S 8 3 3 1 1 62.5

MYCN amplification No 34 15 8 6 5 55.8 0.627 Yes 8 5 2 1 0 37.5 MZF1-uPEP, MZF1 uORF-encoded peptide; MKI, mitosis karyorrhexis index; INSS, international neuroblastoma staging system.

Table S8 YY1 and MZF1 expression in human NB tissues

Total Positive Positive Group YY1 expression P-Value MZF1 expression P-Value number rates (%) rates (%) − + ++ +++ − + ++ +++ Age <1 year 20 11 4 4 1 45.0 0.011 14 3 2 1 30.0 0.001 ≥1 year 22 2 9 6 5 90.9 2 8 8 4 90.9

Differentiation Well differentiated 8 6 1 1 0 25.0 4 2 2 0 50.0 Poorly differentiated 28 7 11 9 1 75.0 <0.001 12 9 7 0 57.1 <0.001 Undifferentiated 6 0 1 0 5 100.0 0 0 1 5 100.0

MKI <200 17 9 5 1 2 47.1 0.037 11 3 2 1 35.3 0.033 >200 25 4 8 9 4 84.0 5 8 8 4 80.0

INSS stages Stage 1-2 14 9 3 2 0 35.7 10 3 1 0 28.6 Stage 3-4 20 2 7 6 5 90.0 0.042 3 5 8 4 85.0 0.025 Stage 4S 8 2 3 2 1 75.0 3 3 1 1 62.5

MYCN amplification No 34 10 12 9 3 70.6 0.140 14 9 8 3 58.8 0.598 Yes 8 3 1 1 3 62.5 2 2 2 2 75.0 YY1, Yin Yang 1; MZF1, myeloid zinc finger 1; MKI, mitosis karyorrhexis index; INSS, international neuroblastoma staging system.

Table S9 Correlation between the expression of MZF1-uPEP, YY1, and MZF1

MZF1 expression Low High R-value P-value MZF1-uPEP expression Low 15 15 -0.471 0.002

High 12 0 YY1 expression Low 23 3 0.643 <0.001

High 4 12 MZF1, myeloid zinc finger 1; MZF1-uPEP, MZF1 uORF-encoded peptide; YY1, Yin Yang 1; Pearson’s correlation coefficient was applied to determine the expression correlation.