Supplementary Table S1 Demographic characteristics of HCC patients

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1 Supplementary Table S1 Demographic characteristics of HCC patients Age (year; mean ± SD) ± Gender (number; %) Male 79 (79%) Female 21 (21%) AFP (ng/ml; mean ± SD) ± Tumor encapsulation (number; %) Absent 61 (63.5%) Present 35 (36.5%) Cellular differentiation by Edmondson grading I-II 46 (47.9%) III-IV 50 (52.1%) Tumor microsatellite formation (number, %) Absent 45 (46.9%) Present 51 (53.1%) Tumor size (cm; mean ± SD) Length 8.3 ± 5.0 Width 7.4 ± 4.3 Venous invasion (number; %) Absent 42 (43.3%) Present 55 (56.7%) Direct liver invasion (number; %) Absent 53 (60.2%) Present 35 (39.8%) ptnm staging (number, %) I-II 37(38.1%) III-IV 60 (61.9%) Hepatitis B surface antigen (number; %) Absent 22 (23.4%) Present 72 (76.6%) Background liver disease (number; %) Normal 7 (7.2%) Chronic hepatitis 36 (37.1%) Cirrhosis 54 (55.7%)

2 Supplementary Table S2 Primer sequences used in qrt-pcr study Genes Human NDUFA4 Human NDUFA4L2 Human NDUFA4L2 HRE-1 (ChIP) Human NDUFA4L2 HRE-2 (ChIP) Human 18S Human ACTB Human HPRT Sequences Forward: cagatcatcggtcaggccaa Reverse: tctgtcccaacaaacatctgga Forward: aaaagacatccggggatcat Reverse: tccgggttgttctttctgtc Forward: ctcctccactgcccactcta Reverse: gtcactctgtactctgcctt Forward: gagagcaggtccccagaga Reverse: ctccttggcccagacttatg Forward: gaggatgaggtggaacgtgt Reverse: agaagtgacgcagccctcta Forward: gcactcttccagccttcctt Reverse: aatgccagggtacatggtgg Forward: ctttgctgacctgctggatt Reverse: ctgcattgttttcggagtgt

3 Supplementary Table S3 The target regions of shrna and sirna constructs shrna clones Genebank Target nucleotides shndufa4-30 NM_ shndufa4-88 NM_ shndufa4l2-90 NM_ shhif-1α NM_ ,123-2,141 shhif-2α NM_ ,992-2,012 sirna clones Genebank Target nucleotides sindufa4l2-1 NM_ sindufa4l2-3 NM_ sindufa4l2-4 NM_

4 Supplementary Table S4 Clinico-pathological correlation of NDUFA4L2 over-expression in human HCC Clinico-pathological features No. of Cases Mean P value Tumor size 5 cm > 5 cm Cellular differentiation by Edmondson grading I II III IV Tumor encapsulation Absent Present *0.039 Tumor microsatellite formation Absent Present *0.002 Direct liver invasion Absent Present Hepatitis B surface antigen Absent Present Venous invasion Absent Present *P<0.05; N=100

5 Supplementary Table S5 Down Regulated Genes in NDUFA4L2 Knockdown HCC cells Gene NTC (FPKM) shl2-90 (FPKM) Fold change SH3GL1P ADAM SCAMP1-AS SH3YL HIST1H3E REREP NR3C SEC31B MYO7B TMEM254-AS C21orf EXOC3L PRH KLF DENND2C MAGEC C9orf CHSY MRC RPEL UNC5B-AS KLF3-AS DNAAF LRRC AHRR COL11A C1QL KCNMB NFATC NECAB CCDC FAM160A FAM27E LDLRAD APOH HSD11B LOC PTPN GPR RXFP RHD SPDYE7P ENO1-AS LINC HIST1H3J HIST1H2AH LOC SPINK LINC RBAKDN LOC LINC

6 CHKB-AS LOC CLLU1OS LOC LOC LOC CSAG LOC ATP6AP1L LOC RASSF1-AS KCNE ORM LINC CLEC3B LOC C1orf TAS2R LY6G6C ACOT LOC CALML RAB MTNR1A LINC SUGT1P C11orf IL MRGPRE LOC CACNA1G-AS UBAP1L LOC FLJ LYRM CHAD SOWAHD FAM19A GPR LOC LOC ALKBH3-AS PCDHGA BIN3-IT OVCH LOC SCOC-AS ZNF LOC DLX STK4-AS FAAH LINC CHRNA

7 C19orf LOC SLC16A WNT ROBO GNN GPR BEGAIN PDC TMOD LOC C4B_ LOXL NEURL1B LOC NBPF CDKN2B-AS PDE5A SIRPB MRPL42P YPEL RORC RAET1E-AS SNHG OAZ ZNF GDPD OSCP FYN ARMC JAZF DRD GKAP HOXC UCN LINC USP27X-AS PRODH GUSBP AGER ANKRD TMEM200B LINC NAPSA PCDHGB MOB3B RYR SULT2B MARCH C7orf GPR FGF IQUB SLAIN

8 LOC ZMYND PLXNC CYP4V RFESD INHA CA AKAP STAM-AS ID ELFN CLCNKB TP PCDHGC SOX2-OT ENDOU LOC KCNQ TSC22D1-AS C6orf DFNB TMLHE-AS TMLHE-AS EFCAB TNFRSF10C MAP6D HIST1H2BF TMEM255B SEPSECS-AS RDH CFI GCNT GRIN3B LINC LOC SLC25A LEPREL MEF2BNB-MEF2B SLC46A CACNA1I C8orf FRMPD2P CAHM SCN4B LRRC CSTA OCLM CHRD CARD DYX1C HTN FAM177B ESRRG ACAP

9 LOC EDN CD SP LOC ADAM TAS1R EYA4-AS LINC ABCA NELL CD27-AS HELB STRC LOC GK3P KHK TMEM TPRG BCO CNTNAP MINOS1-NBL TRIM C3orf RAB RPL21P MMP SLC2A FAM218A IQCH-AS PNMT TMEM SLC46A RIPPLY WWOX HOXC-AS LOC SCGB1B2P LOC LOC C1QTNF MIOX LOC C2orf KRT KEL DCAF13P PCDHA ELN CFP TMEM BDNF-AS FAM47E PCDHGA

10 MIR31HG LMO7-AS FLJ F KLHL LOC LOC HCG IL36G KLHDC LOC PRSS KLHL ASTN ZNRD1-AS PLCXD PLA2G C9orf RGS LINC MX SEPT SPEF TMPRSS PTPRN DHX PADI ICA1L GSTM SIGLEC RTBDN LEF ELAVL RPL36A LOC CDK ANKRD SGK CD C1QTNF3-AMACR FHIT SEMA3B-AS PHGR LINC LOC LOC C2orf IGFL LOC ZRANB2-AS HSPB EPN2-IT CGB SMIM

11 RAET1G TMEM GJC KRTAP TMIGD ARID3C ARGFXP SPATA MS4A GATA PLET CERS LOC ADHFE PLD KIF9-AS TMEM FSIP OLAH VWA5B VWDE GLS SRPK FOLR HTN SPEG FAM90A PLA1A SLC25A21-AS ATP2C SLITRK UGT1A LOC TMEM LINC FOXD4L GPR ANKS1B TRAF3IP BHLHE40-AS NLRP EGFL NFATC ERICH HGF NAALADL CCDC KRTAP KCNIP LOC POU6F PCDHGB ARHGAP NPM

12 SH2D3C RHCG MAF ULK4P PPEF PRKCZ TBC1D10C LOC LOC PTTG3P CALY TAS2R GNMT APOOP SPATA LINC TAS2R TTLL TTTY DLEU2L APOF PPM1J PROC ACTG1P KLRG PIPOX ABHD12B PTH1R MEIOB NBPF25P LOC GUSBP LINC LOC DNASE1L ALX LRRC SAMD LOC SLC13A LOC APOBEC VN1R GP1BA LOC CBR3-AS NRN1L CCL UPK LINC CATSPER NAA LOC SPNS

13 LOC FUT PALM PPIEL KLHL7-AS FAM69C UGT1A LINC KIAA NBPF LCN WWTR1-AS LOC SLC46A GUCY2EP UNC5A CRHR PRR TPTE LOC A4GNT HCK CFHR AIFM AGR IGF AMIGO CTD-2201I THBS LOC SAMD12-AS C1orf N4BP2L MATN ACRBP C21orf PAOX NDUFA4L EIF3CL EIF3CL PHOSPHO2-KLHL BLOC1S5-TXNDC TBC1D3H TMEM256-PLSCR GPRASP E TMEM189-UBE2V KRTAP E E-05 FAM156B E E-05 BLOC1S1-RDH E E-06 HLX-AS E E-06 RAD51L3-RFFL E E-06 LOC E E-07 MIA E E-08 ZNF559-ZNF E E-12

14 PCNA-AS E E-15 FKBP1A-SDCBP E E-25 LOC E E-35 URGCP-MRPS E E-36 STX16-NPEPL E E-84 DEAR E E-151 APOD BEST BEX BPIFA C10orf C11orf C17orf C5orf66-AS CAPZA CARD CASQ CD CDRT CECR5-AS CGB CGB CLEC7A CRYAA CSN1S2AP CST CXorf CXorf CYCSP CYP46A DEPDC1-AS EFHB EFHD ERAS FAM83C-AS FLJ FLJ FLJ GHRL GJB GLUD1P HCRT HIF1A-AS HIST1H2BM HIST1H2BO HIST1H3A HIST1H3G HOXC-AS HYPM IGSF KLHDC KRTAP KRTAP LINC

15 LINC LINC LINC LINC LINC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LOC LYG MGP MIR MIR MOGAT MT1L MUCL NAPSB NKG NLGN4Y-AS NTF OR52N OR5E1P OVOL1-AS OVOL PCAT PCP4L PLA2G4D RAB33A RETNLB RFTN RNASE RPS6KA2-AS SALL SCGB2A SCGB3A

16 SFTPC SLURP SNAR-A SNAR-A SNAR-A SNAR-A SNAR-A SNAR-A SNAR-A SNAR-A SNAR-A SNAR-A SNAR-A SNAR-A SNORA SNORA SNORA SNORA74B SNORA7B SNORA SNORD SOCS2-AS STATH TAC TEX TGIF2LY TMEM TMEM TMEM TMEM TNP TOMM20L TRERNA UBD UBE2NL VPREB WFDC

17 Supplementary Table S6 Up Regulated Genes in NDUFA4L2 Knockdown HCC cells Gene NTC (FPKM) shl2-90 (FPKM) fold change OR2J Inf PYY Inf RNASE Inf CEACAM Inf ACTA Inf LOC Inf FCRLA Inf HSD Inf LOC Inf SYNJ2-IT Inf C16orf Inf SAA Inf RSPH Inf LOC Inf LOC Inf EVA1A Inf LINC Inf LOC Inf PGC Inf LINC Inf LOC Inf WFDC Inf LIPF Inf LINC Inf SPANXN Inf TAS2R Inf LOC Inf SPINK Inf NBEAP Inf CD200R1L Inf LGALS Inf PYDC Inf LHB Inf LOC Inf AANAT Inf PPP4R Inf TTTY Inf CXorf Inf FGF Inf PRAC Inf LTB Inf SPERT Inf LOC Inf C9orf Inf MMP Inf OPA1-AS Inf LINC Inf CLDN Inf CITED Inf LOC Inf LPP-AS Inf CHI3L Inf

18 GPR Inf LINC Inf TNFRSF Inf LOC Inf LOC Inf HIST1H3I Inf LOC Inf LINC Inf CCER Inf CST Inf CYP11A Inf DNAJB5-AS Inf CASP Inf ECSCR Inf C2orf Inf LINC Inf STH Inf LOC Inf LOC Inf PPP1R14A Inf NUCB1-AS Inf KRT Inf B3GNT Inf LINC Inf MT1A Inf MT1G Inf C6orf Inf DOCK9-AS Inf SPARC Inf KLRC Inf TAS2R Inf EIF1AX-AS Inf TNKS2-AS Inf AWAT Inf LOC Inf XCL Inf CYP2C Inf NEBL-AS Inf C1orf Inf PAGE2B Inf LOC Inf CLRN Inf TAL Inf PPIAP Inf HIST1H3F Inf RIIAD Inf HIST1H2BL Inf LOC Inf LOC Inf LOC Inf SCARNA Inf PINLYP Inf CPSF4L Inf APOC Inf

19 LOC Inf LOC Inf PSPN Inf GNG Inf SNORA Inf SNORA59A Inf SNORA59B Inf SNORA70G Inf SNORD Inf SNORA71C Inf MIR Inf SNORA14B Inf SNORA Inf SNORA Inf SNORA Inf MIR Inf KCNRG 2.78E E+87 RFPL3 1.30E E+34 SPECC1L-ADORA2A 5.49E E+29 DNAJC25-GNG E E+25 MIA-RAB4B 5.14E E+19 SNORA E POC1B-GALNT4 2.79E FAM27A 5.90E SPTY2D1-AS1 4.78E HSPB2-C11orf E INS-IGF2 2.19E LOC E IPO11-LRRC C15orf38-AP3S FAM45A LOC FAM72D GSTT GSTT COMMD3-BMI JMJD7-PLA2G4B CCDC LOC SERPINA PPP1R14D AGT SLAMF IBSP SLC5A LCN C14orf DNAJB LOC BZRAP1-AS UCN PPAN-P2RY RMRP APOBEC3G

20 FAM157B LOC S100A DACT3-AS CTSE DGCR TM4SF KLHDC7B GBP DKK GGT C1orf PPP1R1B LOC CYP2D ENPP LSMEM TUSC CD DCAF4L SOX NAAA CXCL PRR15L DNER UCA CCAT APOA PLCB SUGT1P LOC CELP NR0B C6orf DUSP TNRC18P ISM TNFRSF GPBAR LYZ CYP2C C3orf ZNF OSGEPL1-AS RGPD ACKR SELPLG DISP PAQR SERPINA GLI ROS HIPK1-AS RRAD

21 FLNC ENG CD SNORD LY6G5C TSACC LINC LINC AKR7A2P TMEM ZNF LINC ZNF585B NBPF25P CDHR MIR497HG SLC37A C14orf CYP24A MGAT5B TCTEX1D DSG ROR FXYD DUOXA CCDC IL C1QTNF CCDC28B LOC GPR DIRC NBPF RPPH RFPL3S SLCO1B EPHB FAM83A APOE TAPSAR ARHGEF RGPD MYL FLT3LG C20orf LOC FAM209A LOC RBPMS-AS LOC DLEU LINC C11orf ACOT

22 FLJ EPCAM MAN1C MCF2L AVPR HPN-AS STXBP RGS STMN EHF APITD1-CORT CD DEFB TUBA4B GFPT FGG FAM46B CCDC CXCL C14orf RAB3C CD79A TINAG TMEM86A LINC ZDHHC NARR LOC HTRA CPLX FAM71F TNNT LOC NFE SOCS GALR BPIFB NTN STAU2-AS CACNG CCDC LOC RFTN FGFR APOB NKD CD FMNL SHISA FAM222A FRMD CYTIP IHH MYO1F

23 ICAM RGPD ITGB MFAP3L FAM225B OLFM AUNIP CDC25A GNRHR SLC51B CHRM ETNK VASH TNFRSF MAP1A C10orf32-ASMT PRICKLE TEK LINC CASP CSF MTTP ATP6V0D PYCRL ADAM FAM47E-STBD SULT1A UNC13A PTPRS DUSP LOC TRIM TMIGD MRGPRF AOAH-IT IGFL PNPLA HS1BP3-IT PPP1R SLC9B PLAC8L GPHA COX7A TYRO3P KRTAP NBPF18P TCAP ENHO USP2-AS CLDN ZDHHC GNAT THAP7-AS LRRC

24 PRSS LOC LINC EP400NL PRR C9orf OLFML HMGN2P SH3PXD2A-AS MINOS1P ELMO TAS1R KLHDC7A MUSK SCEL SLC43A SCG C1S C10orf C15orf ATP8B5P ANKRD33B SRRM2-AS LOC SLC16A LCN C8orf44-SGK ROPN1L SAP30L-AS PMS2P RNF219-AS SENP3-EIF4A ABCB SBK OR7E12P CHAC OTUB KRT IGSF TICAM RPS10P ACTR3C ITGB GCSAML LYVE NPHS LONRF ADAMTS FGL CEACAM ELMO RBM14-RBM SULT1A LIPE

25 IGFBP TSGA PID UGT2B CCR KCNB CACNG SLCO2A ZFHX LINC POLR2J FAM27E CISH SEMA6B KLF TF TNFRSF1B LOC SNHG FKBP IDH1-AS ECM C9orf COPZ LRRC ARL CD3G DGAT2L KRT F JAKMIP ZNF TNS PVRL UNC13D TRIM TAT ABCA GRM UFSP HSF2BP LOC LRRC AMOTL SIRPA C16orf LHX CC2D2B IL4I GLIPR CCDC SKP1P OAF NRARP

26 WWC BIN WNT7B SHANK CD SERPINA RAB27B FER1L RASSF NUAK TCF LOXL TMEM88B PFKFB HBA C6orf SLC7A LINC SNHG LOC LOC LOC ERICH OR1F CETP PSMB RXFP S100A ALX SOX LINC CEACAM ZBTB LOC HOXD HBEGF ZNF PROSER2-AS DUOX APOM SLC43A COL17A FAM225A G0S EGOT SCARA CEBPA KCNE CPLX PRAP OTUD7A USP HIST1H2AI RPRM

27 LAG LOC AIM1L RARRES GCKR CACNA2D NES LGR PPP1R1C EDAR KBTBD SPOCK ITGAM NOS1AP SEPT5-GP1BB INHBB PLA2G4C PAPLN CX3CL LOC P2RX CCL PRKCH MMP KNG VILL C15orf FMNL TAGLN MIR FAM89A ST LAMA DLG3-AS HBA TTC9B LOC POU2F NLRC EVI2A TGFBR3L WDR C15orf PCDHGB8P CEL LOC LOC RTN4RL ASPHD C10orf DLGAP C4BPB ACTR3B PDZK1IP

28 PTGIS TRIML MMRN ARSB ACOX HHIPL CLDN CXCL RGPD TRPM EP300-AS LINC FABP BATF SLX1A-SULT1A STK CTRL SULT1C2P LRG TNFAIP8L THNSL LOC SRGN LGR TTYH RBP TAS2R REP IL1B YPEL NAP1L NIPAL RGS BGN FAM155B C1orf UBAC2-AS SLCO2B IL18R NME1-NME C9orf KIAA WDR BMP PAQR LINC LCA5L IGLON LHX PRTFDC LOC CSF2RA CSF2RA MMP

29 F C LOC PLLP MMP TRPM VGF ABCC6P ZBTB C2CD4D TAS2R TCN GMDS-AS HSPA FUZ PIK3R C10orf NBPF HAUS LOC BTG PARGP XCL ZCWPW LINC LINC SP SLC4A FBXO SPOCK ZNF CYB5A TMEM FAM105A TGM ABCG CRABP

30 Supplementary Table S7 Pathway Enrichment Analysis of NDUFA4L2 Knockdown HCC cells Down-regulated genes Annotation Cluster 1 Count P_Value GOTERM_BP_ALL regulation of membrane potential E-05 GOTERM_BP_ALL membrane depolarization E-04 GOTERM_BP_ALL cellular ion homeostasis E-04 GOTERM_BP_ALL cellular chemical homeostasis E-04 GOTERM_BP_ALL ion homeostasis E-04 GOTERM_BP_ALL regulation of system process E-04 GOTERM_BP_ALL cellular homeostasis E-03 GOTERM_BP_ALL chemical homeostasis E-03 GOTERM_BP_ALL regulation of neurotransmitter transport E-02 GOTERM_BP_ALL regulation of biological quality E-02 GOTERM_BP_ALL regulation of synaptic transmission E-02 GOTERM_BP_ALL regulation of transmission of nerve impuls E-02 GOTERM_BP_ALL regulation of neurological system process E-02 GOTERM_BP_ALL homeostatic process E-02 GOTERM_BP_ALL cellular metal ion homeostasis E-02 GOTERM_BP_ALL metal ion homeostasis E-02 Annotation Cluster 2 Count P_Value GOTERM_BP_ALL multicellular organismal process E-04 GOTERM_BP_ALL tissue development E-04 GOTERM_BP_ALL system development E-03 GOTERM_BP_ALL multicellular organismal development E-03 GOTERM_BP_ALL anatomical structure development E-03 GOTERM_BP_ALL developmental process E-03 GOTERM_BP_ALL organ development E-02 GOTERM_BP_ALL cell differentiation E-02 GOTERM_BP_ALL cellular developmental process E-02 Annotation Cluster 3 Count P_Value GOTERM_BP_ALL cell adhesion E-02 GOTERM_BP_ALL biological adhesion E-02 GOTERM_BP_ALL cell-cell adhesion E-02 Up-regulated genes Annotation Cluster 1 Count P_Value GOTERM_BP_ALL response to external stimulus E-13 GOTERM_BP_ALL response to wounding E-09 GOTERM_BP_ALL inflammatory response E-08 GOTERM_BP_ALL defense response E-07 GOTERM_BP_ALL response to stress E-06 GOTERM_BP_ALL response to stimulus E-06 Annotation Cluster 2 Count P_Value GOTERM_BP_ALL defense response E-07 GOTERM_BP_ALL immune system process E-05 GOTERM_BP_ALL immune response E-04 Annotation Cluster 3 Count P_Value GOTERM_BP_ALL chemotaxis E-06 GOTERM_BP_ALL taxis E-06 GOTERM_BP_ALL locomotion E-03 GOTERM_BP_ALL cell chemotaxis E-03 GOTERM_BP_ALL locomotory behavior E-03 GOTERM_BP_ALL behavior E-03

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