SUPPLEMENTARY INFORMATION Supplementary Materials and Methods Cell culture conditions My-La, SR-786, HuT 78 and HH were grown in RPMI 1640 medium (Sigma Chemical, St Louis, MO, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin. DERL-7 was grown in RPMI 1640 medium supplemented with 20% fetal bovine serum, 1% penicillin/streptomycin and 20 ng/ml interleukin 2 (PeproTech, Rocky Hill, NJ, USA). MJ was maintained in IMDM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were maintained at 37 C in a humidified 5% CO 2 incubator. Antibodies The following primary antibodies were used for immunoblotting: rabbit pab NIK (#4994, Cell Signaling, Beverly, Massachusetts, USA), mouse mab NF-κB p52 (#05-361, Upstate Biotechnology, Lake Placid, NY, USA), rabbit pab NF-κB p50 (nb600-1207 Novus Biologicals), mouse mab α-tubulin (Sigma-Aldrich Inc., St. Louis, MO, USA), rabbit pab α-tubulin (ab15246, Abcam plc, Cambridge, UK), rabbit pab cleaved caspase-3 (#9661, Cell Signaling), rabbit pab IKK alpha (#2682, Cell Signaling) and rabbit pab IKK beta (#2684, Cell Signaling). Detection was carried out using fluorescent-labeled secondary antibodies (Alexa 680 and Alexa 800, Rockland, Gilbertsville, PA, USA) and an Odyssey Infrared System Scanner (LI-COR Biosciences, Lincoln, NE, USA). For immunohistochemistry, antibodies and conditions are summarized in Supplementary Table S1.
Quantitative RT-PCR For NIK (MAP3K14) detection, forward primer 5 -GGCCTGGGAAAGTCCTTG-3 and reverse primer 5 - CTGCTCCAGACATCCACCTT-3 were used with universal probe #17 (Roche Diagnostics) and TaqMan Universal PCR Master Mix (Applied Biosystems). Human GUSB and 18S FAM probes (Applied Biosystems) were used as endogenous controls in samples and cell lines. For the expression analysis of human IL6, IL21, KLF2, NFKBIA, and CFLAR, the following primers were used with SYBR Green PCR Master Mix (Applied Biosystems): IL6 forward 5 -GAAAGGAGACATGTAACAAGAGT-3, IL6 reverse 5 - GATTTTCACCAGGCAAGTCT-3, IL21 forward 5 - AGCTGAAGAGGAAACCACCTTC-3, IL21 reverse 5 - GAATCACATGAAGGGCATGTTAG-3, KLF2 forward 5 - CAAGAGTTCGCATCTGAAGGC-3, KLF2 reverse 5 - TGTGCTTTCGGTAGTGGCG-3, NFKBIA forward 5 -CAACTACAATGGCCACACGTG-3, NFKBIA reverse 5 - GCAGTCCGGCCATTACAG-3, CFLAR forward 5 -CTGATGGCAGAGATTGGTGAG-3, CFLAR reverse 5 - GGCAACCAGATTTAGTTTCTCCA-3. The gene expression was analyzed in a 7900HT Fast Real-Time PCR System (Applied Biosystems). RNA interference Small interference RNAs (sirnas) against NIK (MAP3K14) or a non-template control (100 nm stealth sirna, Invitrogen Carlsbad, CA, USA) were used to silence NIK in two lymphoma cell lines. Initial studies with five NIK sirnas were performed to select the sequences with the highest knockdown efficiencies. SiRNAs were efficiently introduced into the cell lines by microporation (Microporator MP-100, Digital Bio, Seoul, South Korea). A fluorophore-labeled sirna (siglo Red, Dharmacon Inc., Lafayette, CO, USA) was used to estimate the transfection efficiency, which was greater than 95% in all experiments. The knockdown of IKBKB (IKKβ) and CHUK (IKKα) was performed in the same way. The following stealth sirnas from Invitrogen were used: sinik1 (MAP3K14VHS40827 validated sirna), sinik2 (MAP3K14HSS113310), siikkα1 (CHUKHSS101936), siikkα2 (CHUKHSS174353), siikkβ1
(IKBKBHSS105292), siikkβ2 (IKBKBHSS105294) and sintc (non-target stealth RNAi negative control, 12935-200). Cell viability and cell cycle analysis Both cell viability and cell cycle were assessed in a FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). SiRNA transfected cells were harvested 48, 72 and 96 hours after microporation, washed with PBS and resuspended in 200 μl Annexin V binding buffer (BD Biosciences). Two μl APC-Annexin V (#550474, BD Biosciences) were added to each tube and cells were incubated in the dark for 15-20 minutes. Before acquisition in the cytometer, DAPI (final concentration 10 μg/ml) was added and the viable cell population (DAPI/AnnexinV negative) was quantified using FlowJo software (FlowJo version 7.6.1, TreeStar Inc., Ashland, OR, USA). For cell cycle analysis, cells were collected 24, 48 and 72 hours after sirna transfection, washed with PBS and fixed with chilled 70% ethanol. Before acquisition in the cytometer, cells were washed in PBS and 200 ng/ul RNase A (Qiagen) was added. After 30 minutes of incubation, 10 μl of propidium iodide (P4864, Sigma Aldrich) was added, cells were acquired in the cytometer and data were analyzed with FlowJo. Gene expression microarray hybridization procedure Pellets were collected 48 hours after sirna transfection and RNA was extracted as described above. Three independent experiments were performed and each sample was hybridized onto a separate microarray. Purification, amplification, labeling and hybridization onto 4x44K Whole Human Genome Oligo Microarray were done in accordance with the manufacturer s instructions (Agilent Technologies, Inc., Santa Clara, CA, USA). Shortly, 300 ng of RNA were amplified and sample RNAs were labeled with cyanine 5-conjugated CTP (Cy5). A human universal RNA reference pool (Stratagene, La Jolla, CA) was
concurrently marked with cyanine 3-conjugated CTP (Cy3). After labeling and purification, the incorporation rates of Cy3 and Cy5 were calculated using NanoDrop (Thermo). For two-color hybridization, each Cy5-labeled sample was hybridized onto a separate microarray against the Cy3- labeled reference pool. After hybridization at 65 ο C for 17 hours, slides were washed and scanned in a G2565AA microarray scanner (Agilent Technologies). Feature extraction software (Agilent Technologies, version 10.7.3.1) was used to read and process the microarray images. Lowess intraarray and quantile interarray normalization were applied and statistical analyses were subsequently performed.
Supplementary Tables Table S1. Antibodies and conditions for immunohistochemistry Antibody Species (clone) Company Catalog number Dilution Antigen Retrieval Detection System Instrument Millipore, Billerica, MA, Citrate ph 6 + AUTOSTAINER PLUS, DAKO, p100/p52 mouse monoclonal USA 05-361 1/1000 Proteinase K EnVision FLEX, DAKO Glostrup, Denmark p105/p50 rabbit polyclonal GeneTex, Irvine, CA, USA GTX15485 1/1 Citrate ph 6 EnVision FLEX, DAKO AUTOSTAINER PLUS, DAKO, Glostrup, Denmark RelB rabbit polyclonal Santa Cruz biotechnlogy, Santa Cruz, CA, USA sc-226 1/1250 Citrate ph 6 EnVision FLEX, DAKO AUTOSTAINER PLUS, DAKO, Glostrup, Denmark p65 (RelA) mouse monoclonal (F-6) Santa Cruz biotechnlogy, Santa Cruz, CA, USA sc-8008 1/350 Citrate ph 6 EnVision FLEX, DAKO AUTOSTAINER PLUS, DAKO, Glostrup, Denmark c-rel rabbit polyclonal Calbiochem, Darmstadt;Germany PC139 1/400 Citrate ph 6 EnVision FLEX, DAKO AUTOSTAINER PLUS, DAKO, Glostrup, Denmark Table S2. Multivariate Cox analysis Results from a multivariate Cox regression analysis considering NF-κB and main clinical features in PTCL (Age >60, extranodal sites >1, elevated serum LDH levels, ECOG performance status >1 and Ann Arbor stage >2). LDH = Lactate dehydrogenase, ECOG = Eastern Cooperative Oncology Group, IPI = International Prognostic Index, CI = Confidence Interval. Variable p-value Hazard ratio (CI 95%) NF-κB 0.047 2.145 (1.009-4.560) Age 0.170 1.860 (0.767-4.511) Extranodal sites 0.970 1.018 (0.401-2.585) LDH 0.012 2.890 (1.268-6.591) ECOG 0.451 1.329 (0.635-2.783) Stage 0.993 0.996 (0.383-2.587) Variable p-value Hazard ratio (CI 95%) NF-κB 0.065 1.820 (0.964-3.438) IPI 0.016 1.378 (1.061-1.790)
Table S3. Immunohistochemical staining of NF-κB in T cell lymphoma cell lines Evaluation of the expression of the different NF-κB members by immunohistochemistry in paraffinembedded cell lines. The expression was categorized into strong (+++), medium (++), weak (+) or negative (-), based on the nuclear localization. p52 RelB p50 c-rel p65 My-La +++ + +++ + + SR-786 +++ + +++ ++ + HuT 78 ++ ++ +++ + + DERL-7 - - +++ +++ +++ HH +++ + +++ +++ ++ MJ +++ ++ +++ +++ ++
Table S4. Pathways positively correlated with NIK expression in PTCL Pathways correlated with NIK expression in PTCL samples. Gene sets correlated with NIK expression in a series of 37 PTCL samples, analyzed by Gene Set Enrichment Analysis, are presented (FDR<0.15). NES = Normalized Enrichment Score, FDR = False Discovery Rate, SIZE = number of genes included in the gene sets. NAME NES FDR q-val SIZE CD40 SIGNALING DURING GC DEVELOPMENT 1.688 0.090 159 IL1R PATHWAY 1.677 0.091 31 NFKB TARGET GENES 1.642 0.093 111 EXTRINSIC APOPTOSIS 1.690 0.097 78 IL2 PATHWAY 1.644 0.098 23 TALL1PATHWAY 1.651 0.101 15 TNFR2 PATHWAY 1.934 0.102 18 T CELL CYTOKINE SIGNALING 1.690 0.106 158 BLIMP-1 TARGETS 1.601 0.108 153 APOPTOSIS SIGNALING 1.614 0.109 109 B-CELL ANERGY 1.605 0.111 22 DEATH PATHWAY 1.693 0.112 31 TCR PATHWAY 1.737 0.118 43 PI3K/AKT SIGNALING 1.575 0.120 34 NKT PATHWAY 1.564 0.121 27 MAPK PATHWAY 1.697 0.123 86 JAK-STAT SIGNALING PATHWAY 1.847 0.133 82 IL2RB PATHWAY 1.698 0.139 35 BCELL-TCELLCELL CALCIUM SIGNALING 1.738 0.140 140
Table S5. Gene Sets enriched in control cells after NIK knockdown in MyLa and SR-786 cells Gene sets significantly enriched (FDR<0.10) in the control cells (downregulated in sinik cells) using Gene Set Enrichment Analysis to compare the expression profiles of control and sinik cells. NES = Normalized Enrichment Score, FDR = False Discovery Rate, SIZE = number of genes included in the gene sets. MYLA SR786 Gene Sets enriched in control cells Gene Set NES FDR q-val Size Genes in core enrichment XBP1 TARGETS NFKB TARGET GENES G2M CHECKPOINT REGULATION -2.1333 0.0013 58-1.9772 0.0038 106-1.7573 0.0530 75 IL3PATHWAY -1.6643 0.0937 15 SSR3, RPN1, FTSJ1, ITGAM, SSR1, PDIA4, SPCS3, GLRX, SLC33A1, DNAJC10, SYVN1, LMAN1, TMED10, HM13, PDIA5, ARF3, SEC24C,SPOP, SLC3A2, SEC23B, FKBP14, DDOST, PDIA3, COX15, COPE, PPIB, HERPUD1 ICAM1, BCL2L1, EMR1, SDC4, NFKBIA, HLA-F, IL6, TNFAIP3, CD44, PLEK, STAT5A, VIM, LTA, CFLAR, STX4, SMAD7, CD40LG, BUB1B, PRKCD, CEP110, BIRC3, STAT1, CCL3, IRF1, FNDC3A, CD83, GADD45B, CCND2, REL, FAS, RRAS2, LSP1, ELL2, IER2, MAP3K1, RFTN1, NFKB2, IL10, NFKB1, NCF2, CXCL9 AURKA, KIF2C, SKP2, TOP2A, MDC1, BUB1, CCNH, BUB1B, CUL1, BRSK1, CCNB3, MDM2, CENPA, GADD45B, UBD, BUB3, IRF5, CDC20, PRC1, TTK, ZC3HC1, HMMR, STIL, CDC16, CENPF, CCNF, PRKDC, PLK1, BRCA1, CDC6, CDKN2D, CHEK2, CCNB1, SFN, BIRC5, CDC34, KIF11 MAP2K1, IL3RA, STAT5A, STAT5B, MAPK3, CSF2RB, IL3 Gene Set NES FDR q-val Size Genes in core enrichment XBP1 TARGETS -2.0985 0.0014 58 JAK-STAT SIGNALING PATHWAY -1.8062 0.0419 72 NFKB TARGET GENES -1.7378 0.0563 106 SSR3, ISG20, GLRX, DNAJC10, FKBP14, SLC33A1, SRP54, PDIA4, LMAN1, SPCS3, PDIA3, PDIA5, SPOP, PPIB, SLC3A2, ARF3, DAD1, HSP90B1, RPN1, RHOQ, SEC23B, SEC24C, ITGAM, DNAJB9 IL7, STAT3, IL6, IL26, MAP2K1, IL12RB2, STAT5A, STAT6, IL4, IL15, SOCS3, STAT1, IL23R, STAT5B CD44, ID2, IL6, ICAM1, EMR1, SPI1, STAT5A, BIRC2, TNFAIP3, GADD45B, IER2, CCL3, STX4, STAT1, LTA, RFTN1, NFKBIA, CFLAR, VIM, PLEK, CD40LG, CSF2, BCL2A1, HLA-F, RET, BANK1, IL10, SDC4, JUNB, TNF, BIRC3, NFKB2, PRKCD, NFKB1, SMAD7, FNDC3A, SLAMF7
Table S6. Genes with altered expression after NIK knockdown List of genes differentially expressed (paired t-test) between control and NIK knockdown cells. Genes with FDR <0.05 and log 2 foldchange >±0.6 are shown. My-La SR-786 NAME sinik Average log 2 foldchange FDR NAME sinik Average log 2 foldchange PON2-1.934791056 0.0004736 IL6-1.56822828 0.0003872 YAP1-1.922000964 2.33E-05 IL21-1.342954078 0.0005381 CTH -1.852426204 0.0107025 ENOX1-1.062669055 0.0016482 HOXB9-1.553801996 0.0039192 ZBED2-1.039222523 0.0058156 DDIT3-1.515654823 0.0122935 HYAL4-1.033591094 0.0034573 BTN3A3-1.515302695 5.32E-05 OCA2-1.024535756 0.0002408 RGS16-1.46288644 4.27E-05 COL15A1-1.00557959 0.0013973 LARGE -1.451885883 0.0019536 EDN2-1.003266321 0.0065465 NCRNA00176-1.449751101 0.0002993 IL22-0.939237515 0.0331672 TNFSF4-1.426571007 2.73E-05 ADAM8-0.938545293 0.0061747 ATP8A2-1.418530434 0.001456 OTOA -0.92926982 0.0007472 ADAM8-1.353864258 0.0036287 C7orf68-0.910850386 0.0227028 RAB9A -1.322963403 0.0008235 C6orf105-0.868089965 0.0016482 OTOA -1.320726393 0.0004736 YAP1-0.847047318 0.0016482 EDN2-1.314166818 0.0016865 RILPL2-0.840538613 0.0045345 LOC284561-1.313985454 0.0039758 SQRDL -0.837119086 0.0075789 TIFA -1.254222905 0.0005777 DDIT3-0.828681553 0.0396716 WARS -1.239704966 0.0028519 IL26-0.806261327 0.0017192 IL3RA -1.238680679 0.0002543 PGLYRP4-0.797476697 0.006786 IL6-1.234867676 0.0002993 GFPT2-0.788825076 0.0017192 IL15-1.232436955 0.0010314 CCL17-0.778448958 0.0045345 XBP1-1.222232536 0.0032711 IL4I1-0.7702507 0.0016482 LOC339047-1.21724901 0.0073262 NEURL3-0.758150177 0.0019121 PFKM -1.213776737 0.0003366 CNR1-0.757490474 0.0007587 ATF3-1.212757429 0.0026491 HSD11B1-0.753843833 0.0499396 NFKBIA -1.211331581 0.0002538 IFIH1-0.74215219 0.0034573 IFRD1-1.20960878 0.0035494 SLC2A14-0.732820111 0.0084535 IL21-1.177808692 0.0005299 C10orf116-0.731320788 0.0016482 RAB9P1-1.174504007 0.0010643 BTN3A3-0.718828055 0.0072637 SQRDL -1.16179072 0.0002726 RGS16-0.718265539 0.0332744 OLAH -1.160986553 0.0016632 IL1A -0.714037383 0.0017192 NCRNA00158-1.156707982 0.0013179 MIR155HG -0.712981469 0.006786 IL9-1.1381125 0.0063225 LOR -0.708533824 0.00483 EMR1-1.128732874 0.0010813 PLXDC2-0.698141365 0.0117254 PSAT1-1.119356186 0.0086172 BBS12-0.687269737 0.006786 C6orf105-1.114067568 0.0013179 TNFSF4-0.684512317 0.0068783 GLUD1-1.111437531 0.0018391 EXTL2-0.677975963 0.0016482 RAI14-1.099128605 0.0024024 CYP1A1-0.675064083 0.0014421 ZNF643-1.092170316 0.0025008 EEF1A2-0.674139969 0.006786 JMY -1.07028315 0.0012011 INHBA -0.673591533 0.0034573 IFIH1-1.066138724 0.0022377 MGST3-0.662971712 0.0066029 BBS12-1.06582497 0.0143751 PLEKHG1-0.660587702 0.006786 AMICA1-1.060604387 0.0002993 IL9-0.656046843 0.0260374 C10orf116-1.059101372 0.0002543 TNFAIP6-0.646189058 0.00423 GLUD2-1.054757386 0.0010643 KCNQ2-0.642858878 0.0050549 GSDMB -1.046394403 0.0010021 VDR -0.64099722 0.0034573 PTP4A3-1.023680834 0.0011469 LTA -0.63799439 0.0082572 FHL2-1.020758034 0.0115779 TIFA -0.635856771 0.0088409 ATF5-1.011900573 0.0202009 NRCAM -0.630728316 0.0016482 SH2B3-1.008459399 0.0016319 BTNL8-0.630724422 0.0257024 COL15A1-1.007926246 0.0011876 WARS -0.630327479 0.0274456 PGLYRP4-0.998917849 0.0022377 NFKBIA -0.617999157 0.0042331 GARS -0.994798816 0.0052621 NCRNA00176-0.612952927 0.0137663 PLXDC2-0.993181017 0.0014473 SLC45A3-0.609342116 0.0109341 CLIP2-0.991614696 0.0040966 ATP8A2-0.607971372 0.0072637 LTA -0.991445888 0.003663 IL15-0.605475346 0.0016482 LOR -0.98685254 0.0013179 JMY -0.605412542 0.0155819 CHAC1-0.973489811 0.0111449 BIRC3-0.602605374 0.0023258 RNGTT -0.972163309 0.0041243 DAZAP2 0.605739515 0.0068783 DDX4-0.968590392 0.0005191 TMTC2 0.619221413 0.0135259 STRA6-0.967498798 0.0018169 CCR5 0.629018019 0.012784 FERMT2-0.964713396 0.0027313 CDKN2C 0.632636947 0.0177926 MARS -0.959201131 0.0040966 CARHSP1 0.647465842 0.0458475 GFPT2-0.955772933 0.0002187 SLC9A9 0.654404325 0.0130454 CCL5-0.954297766 0.0001355 POU2AF1 0.665644763 0.0114545 SLC26A9-0.951068933 0.0017489 BFSP1 0.667680797 0.0065434 SERPINB8-0.94727664 0.0010195 MGC42105 0.684572714 0.007964 IL1R2-0.943173326 0.001144 GLCCI1 0.685682887 0.0084496 HYAL4-0.94234633 0.0037085 TRBV5-4 0.687819738 0.0075631 CARS -0.937493141 0.0072454 UTS2D 0.691955342 0.0093629 TRERF1-0.933651214 0.0053108 LGR4 0.713719116 0.007964 SHMT2-0.933235146 0.000625 SLC26A11 0.733509574 0.0094512 DARS2-0.931511905 0.0077505 PIM2 0.734472078 0.006786 FDR
ZG16B -0.931143664 0.0054628 SLC39A10 0.752225968 0.006786 HLA-F -0.930453169 0.0006903 SLC38A6 0.768399527 0.0045345 GPT2-0.930128563 0.0137405 IRS1 0.769230272 0.0132315 PCK2-0.928588691 0.0067871 KCNJ12 0.770534035 0.0072483 GAS7-0.925309014 0.0004088 RASGRP3 0.807073503 0.0068783 MAP2K1-0.919939572 0.0003541 NME7 0.807688627 0.0016482 BEST1-0.911724862 0.0382266 ENPP2 0.818914222 0.0007587 IL1A -0.910130859 0.0003366 SAMD9 0.836868535 0.0034573 FAS -0.906938033 0.0024666 RTP4 0.863727994 0.0053112 SLC1A5-0.906544899 0.0037371 GPR15 0.921759833 0.0122173 DDR2-0.906034106 0.0022377 ARNT2 0.935200017 0.0016482 VDR -0.905869685 0.0027011 SAMD9L 1.064648678 0.0013973 ICAM1-0.892159479 0.0003343 ENC1 1.099658373 0.0028822 PLEKHG1-0.890848754 0.0011722 IGHM 1.12886267 0.0003872 ZBED2-0.886969129 0.0034927 DNAH12 1.144217805 0.0006028 UBD -0.880563319 0.0341441 KLF2 1.21654953 0.0016482 CCDC50-0.877866954 0.0116585 F2R 1.335973098 0.0007472 RHEBL1-0.877850177 0.0005062 FAM9B 1.505830969 0.0034573 CD302-0.874078413 0.0003541 HTR2B 1.663156191 0.0016482 PSME2-0.874058279 0.0004736 PMCHL1 2.37011489 0.0005635 FSCN1-0.871712221 0.0002993 PMCH 3.062090421 0.0003872 CD44-0.861345811 0.0022377 CCNB1IP1-0.860559982 0.0024024 INSIG1-0.85723793 0.0020418 INHBA -0.850571462 0.0013179 TNFAIP6-0.850042801 0.0011288 MIR155HG -0.843729348 0.0005395 FCER2-0.829975952 0.0032711 TTC25-0.82830987 0.0258457 C7orf68-0.818436699 0.0069969 SGCA -0.817955416 0.0078615 TUBE1-0.817628718 0.0066864 TJP2-0.81350072 0.0079871 ERMN -0.812661423 0.0013179 GLT25D2-0.808327253 0.025341 IGSF3-0.804935478 0.0083197 FDFT1-0.804700996 0.0039192 PSMB9-0.802283468 0.0004433 MID1IP1-0.797817137 0.0037743 DHCR7-0.796549659 0.0014473 FKBP11-0.794344302 0.0040596 RAD23A -0.794168905 0.0039758 DNAJC5B -0.791321612 0.0104295 KIF21B -0.788292652 0.0056782 EHD4-0.788181309 0.0048336 IARS -0.787947996 0.0126866 GBE1-0.786464908 0.014188 RILPL2-0.785413947 0.0016066 AARS -0.785250246 0.009338 LOC374491-0.785184904 0.0361809 SARS -0.781620221 0.0141326 CHRNA6-0.779720214 0.0069287 EIF4EBP1-0.777059662 0.0072114 B4GALT4-0.775724415 0.0006144 PSME1-0.774929902 0.0017334 ST8SIA1-0.773339783 0.0010021 BIRC3-0.771507535 0.0007483 GEM -0.771060645 0.0013276 ALPK1-0.770627018 0.0036287 CLDN16-0.769291738 0.001387 LONP1-0.767648855 0.0128013 CNR1-0.761875095 0.0005209 NFKBIE -0.761154885 0.0039816 DHRS2-0.759217748 0.0008235 WIPI1-0.752369241 0.0196245 LGTN -0.750018643 0.0067299 MYO5C -0.748768431 0.0071926 YARS -0.748473207 0.0053956 FASN -0.746867398 0.0010387 SLC7A11-0.746477175 0.0358324 KCNH8-0.743820883 0.0124797 DNAJC7-0.734592598 0.0078722 LTB -0.731316498 0.0458565 RASGRF2-0.730403289 0.0075461 KDM4A -0.730177277 0.0074789 SESN2-0.726326423 0.0044458 SNHG5-0.718291219 0.0088546 ENOX1-0.716172405 0.0032711 EXTL2-0.715309972 0.001456 ADARB2-0.713622523 0.0032715 RUNDC3B -0.709965569 0.0111495 BTNL8-0.707095926 0.0353685 IL18R1-0.706603289 0.0029852 ITLN2-0.704288656 0.0033053 PRR5L -0.704166388 0.0011902 VIM -0.703592866 0.0342568 ASNS -0.703365699 0.0208467 C10orf10-0.703023496 0.0058747
BTN3A1-0.701914384 0.0023107 SLC39A9-0.699935068 0.0122032 GNPDA1-0.697670084 0.0024135 PYCR1-0.695853925 0.0010314 KIAA1147-0.693735431 0.0006802 FKBP14-0.692398035 0.0006625 IL23R -0.687711574 0.0075383 CFLAR -0.682372456 0.0005777 LPIN2-0.678424823 0.0024135 UPK1B -0.678000037 0.0039758 LY75-0.67585202 0.0021176 MGST3-0.673082268 0.0088603 SLC3A2-0.672667919 0.0096626 ACAD11-0.672243108 0.0044099 EEF1A1-0.671387218 0.0157275 TPTE -0.670124466 0.0034077 PLA1A -0.669344481 0.0017542 RFX5-0.666189333 0.0040961 MVK -0.666169222 0.0029733 SEMA4A -0.660833557 0.0358324 HAX1-0.660031962 0.0155398 ATF4-0.658695849 0.0077948 VPS29-0.65411015 0.0170636 FAM122B -0.652317714 0.0149257 DNAJC12-0.651915677 0.0036287 BAZ2A -0.651866685 0.0072212 C20orf112-0.651759708 0.0045246 ACSM3-0.650434395 0.0319709 LIPA -0.64808264 0.0445116 IL4I1-0.647625463 0.0041243 CD80-0.645976845 0.000748 DENND5A -0.645531547 0.0013124 ALDOC -0.641912751 0.0119469 C13orf23-0.641727735 0.0022377 CD83-0.64080144 0.0005299 HLA-H -0.640481314 0.0024135 NPIP -0.639159994 0.0344474 NUDT15-0.636552957 0.0013179 TCAM1-0.633724951 0.0072865 C5orf23-0.6332175 0.014745 LOC645676-0.626964854 0.0022607 GADD45A -0.626408396 0.0218294 GPR87-0.625884378 0.0248351 HERPUD1-0.623979279 0.0106669 APOBEC3H -0.623780948 0.0037937 EPRS -0.622404659 0.0192499 C18orf58-0.621833755 0.031675 CT45A5-0.619897313 0.0023107 MST1R -0.619328701 0.003309 IL22-0.618291265 0.022998 SLC7A5-0.617954778 0.0038632 SCD -0.617722992 0.000867 MAGEB2-0.617664934 0.0034525 BCL2L1-0.616233649 0.000867 CT45A1-0.614843955 0.0032711 KIF2C -0.614517274 0.012767 WDR65-0.614313731 0.004563 PDAP1-0.613319953 0.009614 XPOT -0.611279201 0.0124043 TTC39A -0.60554742 0.0009959 DHRS11-0.605222633 0.0171054 HLA-J -0.605014525 0.0022343 CPNE5-0.603522937 0.0024878 CHST12 0.600478203 0.0058747 IGFBP1 0.60172655 0.0218133 TPSG1 0.602906571 0.030654 F5 0.602917088 0.0204376 MKL2 0.603853391 0.0016865 ZNF579 0.604382561 0.0217079 ASPRV1 0.604430186 0.0024135 OXCT1 0.604514848 0.0018745 PPP1R9A 0.604711916 0.0057866 SFRS6 0.606943004 0.037869 MEGF6 0.607243453 0.0188158 C6orf225 0.60761219 0.0008595 CD7 0.608209326 0.0248351 ABR 0.608637802 0.0008364 AMY1C 0.610960423 0.0128925 C10orf140 0.6119889 0.0026395 CACNA1E 0.614457167 0.0392328 GRK5 0.616979404 0.0033704 MAF 0.61873955 0.0188929 ZMYND11 0.621857534 0.0029388 C18orf50 0.622307323 0.008591 FLJ22184 0.623804502 0.031202 WFDC1 0.625716335 0.0050442 CABYR 0.627694959 0.0034525 DAZAP2 0.628101658 0.0024878
JAZF1 0.628856875 0.0104295 TFPI 0.629113706 0.0028177 SLC19A2 0.630110837 0.0078615 E2F3 0.631246075 0.0057866 LOC100131998 0.632431133 0.0056732 FRAT2 0.632767614 0.0036287 SESN3 0.633478069 0.0143751 ATP6V1G1 0.634302361 0.0008235 PNMA1 0.635962904 0.0010643 HSPA2 0.636440728 0.0121359 CSGALNACT1 0.636869925 0.0058747 UTS2R 0.637660285 0.0155398 ZSCAN10 0.637784327 0.0209157 CDH1 0.638915564 0.0022377 C9orf62 0.639194799 0.0492701 ANKRD13C 0.639758783 0.0117623 TP53INP1 0.640354062 0.0154863 FAM176A 0.641773077 0.0019309 HCN4 0.642817371 0.0175659 ITGB1 0.64383166 0.000867 CDKN1C 0.644657118 0.0273196 NKAIN1 0.645431618 0.028759 CD96 0.646266815 0.0193028 IFI6 0.647130286 0.0121629 RELL1 0.647206775 0.0039758 SH3GL3 0.647566711 0.031675 MTSS1 0.649023397 0.0014473 DEXI 0.649940823 0.0032711 RNF149 0.650464482 0.000748 TMTC2 0.653341183 0.0030616 GPR78 0.653965529 0.0408859 WDR81 0.654587381 0.0052606 LOC338620 0.654851629 0.0014473 PIP5K1B 0.657453343 0.0068517 GRIN2D 0.657751692 0.0221266 GPR153 0.661117936 0.0186212 LOC439951 0.666180275 0.0232916 NCDN 0.668454638 0.0039758 SOX2 0.669649214 0.0057866 HOMER3 0.673607425 0.0004197 FAM116A 0.674688753 0.013925 PM20D2 0.675184652 0.006791 COX6A2 0.675257817 0.0223342 UTS2D 0.677295624 0.0016865 C9orf135 0.678661503 0.0024024 IL7R 0.678668116 0.0052196 C1orf53 0.680504357 0.0018745 GMNN 0.686446624 0.0146245 TIMM8B 0.690258163 0.009776 DKFZP434I0714 0.690663719 0.020726 CACNA1H 0.690809809 0.0077148 KIF25 0.697128815 0.0070761 ITM2A 0.697461077 0.0009888 FAIM 0.698326722 0.0022377 FBXL17 0.699333914 0.0036287 SOBP 0.701633675 0.0057866 ARL4C 0.703526632 0.0014221 PODXL 0.706046918 0.0078615 TNFRSF18 0.714190864 0.001456 IFITM5 0.717669802 0.0237937 KLHDC7B 0.717688854 0.0076972 PPP2CB 0.718978936 0.0010643 VNN2 0.721927409 0.0034927 GPR150 0.722898129 0.0184506 GPR65 0.724629606 0.0093979 ARHGAP12 0.724883946 0.0010387 XYLT1 0.732321045 0.0006903 GPR137B 0.733614882 0.001456 DUX4 0.734823217 0.0170636 ITM2C 0.735973747 0.0024135 HCN2 0.741974509 0.0200838 ENTPD1 0.753504324 0.0007088 LOC644246 0.755710841 0.0130485 RTP4 0.761655691 0.0018745 KCNJ13 0.761733284 0.0003706 ITGB7 0.770385332 0.0037371 CORO2A 0.771035549 0.0052267 NUCB2 0.777044273 0.0218294 CEP70 0.778256455 0.0014473 TTC18 0.778945591 0.0021176 FAM46C 0.782460972 0.0004088 PGBD2 0.784976179 0.0005191 ZNF670 0.788639142 0.0144199 TXNDC3 0.792701325 0.000625 NTNG2 0.808296292 0.0191531 TSHZ3 0.808977421 0.0022377 MUC20 0.81356573 0.0074733 MUC6 0.817343089 0.0312069
SLC26A11 0.821192258 0.0005122 ITM2B 0.823560477 0.001456 SATB1 0.824186699 0.0057107 PSKH1 0.829865305 0.0002993 LIMA1 0.837011078 0.0004101 PTPRC 0.842412639 0.0056732 IRS1 0.842732156 0.0036287 CRABP2 0.847852278 0.0109271 GLCCI1 0.852666646 0.0004433 LAMB1 0.862761816 0.0005299 TULP4 0.864343498 0.0059505 CD28 0.869373338 0.0002993 KCNJ12 0.878525564 0.0119434 LOC541471 0.880262154 0.0039758 SLC44A1 0.899259801 0.0003366 TIPARP 0.903514137 0.0072454 CDKN2C 0.904756655 0.0287771 CASP7 0.934968682 0.0050442 FRY 0.944096922 0.000625 SCG2 0.960531186 0.0013179 RGS9 0.963504858 0.0174748 CCR3 0.963903559 0.0001426 DMBT1 0.970375073 0.0007013 SLC9A9 0.974025772 0.0013179 SAMD9 0.983482396 0.0002083 KIAA1618 0.993904748 0.0032711 SLC7A8 0.99617436 0.0072807 BZRAP1 1.008045363 0.0007332 SLC39A10 1.014493441 0.0048299 HIST1H4A 1.023507021 0.0067714 SLC38A6 1.046761445 0.0009888 GZMB 1.048208195 0.0077555 RBP4 1.088474701 0.006156 BFSP1 1.116554826 0.0002799 TOX2 1.124506227 0.0003706 TRBV5-4 1.14931459 0.0002626 NME7 1.160421501 0.0001431 F2R 1.183383878 0.0001355 GPR15 1.198683418 0.0040961 FAM9B 1.214973133 0.0001431 LOC150759 1.271031346 0.0002939 ENPP2 1.300790508 0.0029852 ENC1 1.318505699 0.00016 IER5L 1.326927321 0.0044371 CCR5 1.379678821 0.0001006 LOC283143 1.415459885 0.0309213 SMCR5 1.466846631 0.0082939 SAMD9L 1.506913189 0.0001872 ARNT2 1.536616704 0.0001355 HTR2B 1.546671664 0.0002385 CFHR3 1.55557288 0.0004088 MGC42105 1.585966236 0.000615 KLF2 1.63901591 0.0002543 ANKRD55 1.641900052 0.0011722 LGR4 1.72804023 2.33E-05 CFH 1.743493205 5.01E-05 PMCHL1 2.355293623 8.00E-07 PMCH 3.077597447 8.00E-07
Figure Legends Figure S1. Overall survival in PTCL-NOS and AITL. Kaplan-Meier curves representing the overall survival times in patients with PTCL-NOS (47 patients, black line) and AITL (30 patients, grey line). No significant difference was observed between the two PTCL subtypes (p=0.48, log rank test). Figure S2. p100 and p105 levels after NIK knockdown. Quantification of p100 and p105 from immunoblots after NIK knockdown in My-La and SR-786 indicate an initial increase of levels of p100 and p105, suggesting a blockage of the processing of these proteins. The data are represented as the mean ±SD of three independent experiments. Figure S3. Cell cycle alterations induced by NIK knockdown. Evaluation of the proportion of cells in each phase of the cell cycle after flow cytometry analysis of propidium iodide staining. No notable changes were observed except for an increase in the number of sub-g1 cells. The data are represented as the mean ±SD of three independent experiments. Figure S4. Knockdown of IKKα and IKKβ in SR-786. IKKα or IKKβ, either separately or together were efficiently silenced in SR-786 cells and the expression of downstream proteins was analyzed by western blot. Figure S5. Efficiencies of NIK knockdown. NIK protein expression after NIK knockdown in My-La and SR- 786 in the three independent experiments performed for the gene expression analysis.
Figure S6. IL6 and IL21 levels in PTCL cell lines. RT-qPCR analysis of the basal expression levels of IL6 and IL21 in PTCL cell lines indicate a high level of expression of these interleukins in NIK overexpressing cells (My-La and SR-786), whereas the expression is almost absent from cells with low NIK levels (HuT 78 and DERL-7). Relative quantities (RQ) are calculated by comparing the expression levels of each individual cell lines with the average IL6 or IL21 levels of the four cell lines.
Supplementary Figures Figure S1
Figure S2
Figure S3 Figure S4
Figure S5 Figure S6