SUPPLEMENTARY INFORMATION
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1 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 (nb 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.
2 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
3 (IKBKBHSS105292), siikkβ2 (IKBKBHSS105294) and sintc (non-target stealth RNAi negative control, ). 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 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
4 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 ) was used to read and process the microarray images. Lowess intraarray and quantile interarray normalization were applied and statistical analyses were subsequently performed.
5 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 /1000 Proteinase K EnVision FLEX, DAKO Glostrup, Denmark p105/p50 rabbit polyclonal GeneTex, Irvine, CA, USA GTX /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 /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 ( ) Age ( ) Extranodal sites ( ) LDH ( ) ECOG ( ) Stage ( ) Variable p-value Hazard ratio (CI 95%) NF-κB ( ) IPI ( )
6 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 HuT DERL HH MJ
7 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 IL1R PATHWAY NFKB TARGET GENES EXTRINSIC APOPTOSIS IL2 PATHWAY TALL1PATHWAY TNFR2 PATHWAY T CELL CYTOKINE SIGNALING BLIMP-1 TARGETS APOPTOSIS SIGNALING B-CELL ANERGY DEATH PATHWAY TCR PATHWAY PI3K/AKT SIGNALING NKT PATHWAY MAPK PATHWAY JAK-STAT SIGNALING PATHWAY IL2RB PATHWAY BCELL-TCELLCELL CALCIUM SIGNALING
8 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 IL3PATHWAY 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 JAK-STAT SIGNALING PATHWAY NFKB TARGET GENES 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
9 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 PON IL YAP E-05 IL CTH ENOX HOXB ZBED DDIT HYAL BTN3A E-05 OCA RGS E-05 COL15A LARGE EDN NCRNA IL TNFSF E-05 ADAM ATP8A OTOA ADAM C7orf RAB9A C6orf OTOA YAP EDN RILPL LOC SQRDL TIFA DDIT WARS IL IL3RA PGLYRP IL GFPT IL CCL XBP IL4I LOC NEURL PFKM CNR ATF HSD11B NFKBIA IFIH IFRD SLC2A IL C10orf RAB9P BTN3A SQRDL RGS OLAH IL1A NCRNA MIR155HG IL LOR EMR PLXDC PSAT BBS C6orf TNFSF GLUD EXTL RAI CYP1A ZNF EEF1A JMY INHBA IFIH MGST BBS PLEKHG AMICA IL C10orf TNFAIP GLUD KCNQ GSDMB VDR PTP4A LTA FHL TIFA ATF NRCAM SH2B BTNL COL15A WARS PGLYRP NFKBIA GARS NCRNA PLXDC SLC45A CLIP ATP8A LTA IL LOR JMY CHAC BIRC RNGTT DAZAP DDX TMTC STRA CCR FERMT CDKN2C MARS CARHSP GFPT SLC9A CCL POU2AF SLC26A BFSP SERPINB MGC IL1R GLCCI HYAL TRBV CARS UTS2D TRERF LGR SHMT SLC26A DARS PIM FDR
10 ZG16B SLC39A HLA-F SLC38A GPT IRS PCK KCNJ GAS RASGRP MAP2K NME BEST ENPP IL1A SAMD FAS RTP SLC1A GPR DDR ARNT VDR SAMD9L ICAM ENC PLEKHG IGHM ZBED DNAH UBD KLF CCDC F2R RHEBL FAM9B CD HTR2B PSME PMCHL FSCN PMCH CD CCNB1IP INSIG INHBA TNFAIP MIR155HG FCER TTC C7orf SGCA TUBE TJP ERMN GLT25D IGSF FDFT PSMB MID1IP DHCR FKBP RAD23A DNAJC5B KIF21B EHD IARS GBE RILPL AARS LOC SARS CHRNA EIF4EBP B4GALT PSME ST8SIA BIRC GEM ALPK CLDN LONP CNR NFKBIE DHRS WIPI LGTN MYO5C YARS FASN SLC7A KCNH DNAJC LTB RASGRF KDM4A SESN SNHG ENOX EXTL ADARB RUNDC3B BTNL IL18R ITLN PRR5L VIM ASNS C10orf
11 BTN3A SLC39A GNPDA PYCR KIAA FKBP IL23R CFLAR LPIN UPK1B LY MGST SLC3A ACAD EEF1A TPTE PLA1A RFX MVK SEMA4A HAX ATF VPS FAM122B DNAJC BAZ2A C20orf ACSM LIPA IL4I CD DENND5A ALDOC C13orf CD HLA-H NPIP NUDT TCAM C5orf LOC GADD45A GPR HERPUD APOBEC3H EPRS C18orf CT45A MST1R IL SLC7A SCD MAGEB BCL2L CT45A KIF2C WDR PDAP XPOT TTC39A DHRS HLA-J CPNE CHST IGFBP TPSG F MKL ZNF ASPRV OXCT PPP1R9A SFRS MEGF C6orf CD ABR AMY1C C10orf CACNA1E GRK MAF ZMYND C18orf FLJ WFDC CABYR DAZAP
12 JAZF TFPI SLC19A E2F LOC FRAT SESN ATP6V1G PNMA HSPA CSGALNACT UTS2R ZSCAN CDH C9orf ANKRD13C TP53INP FAM176A HCN ITGB CDKN1C NKAIN CD IFI RELL SH3GL MTSS DEXI RNF TMTC GPR WDR LOC PIP5K1B GRIN2D GPR LOC NCDN SOX HOMER FAM116A PM20D COX6A UTS2D C9orf IL7R C1orf GMNN TIMM8B DKFZP434I CACNA1H KIF ITM2A FAIM FBXL SOBP ARL4C PODXL TNFRSF IFITM KLHDC7B PPP2CB VNN GPR GPR ARHGAP XYLT GPR137B DUX ITM2C HCN ENTPD LOC RTP KCNJ ITGB CORO2A NUCB CEP TTC FAM46C PGBD ZNF TXNDC NTNG TSHZ MUC MUC
13 SLC26A ITM2B SATB PSKH LIMA PTPRC IRS CRABP GLCCI LAMB TULP CD KCNJ LOC SLC44A TIPARP CDKN2C CASP FRY SCG RGS CCR DMBT SLC9A SAMD KIAA SLC7A BZRAP SLC39A HIST1H4A SLC38A GZMB RBP BFSP TOX TRBV NME F2R GPR FAM9B LOC ENPP ENC IER5L CCR LOC SMCR SAMD9L ARNT HTR2B CFHR MGC KLF ANKRD LGR E-05 CFH E-05 PMCHL E-07 PMCH E-07
14 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.
15 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.
16 Supplementary Figures Figure S1
17 Figure S2
18 Figure S3 Figure S4
19 Figure S5 Figure S6
NES: normalized enrichment score (analyzed using KEGG pathway gene sets in the GSEA software); FDR:
Supplementary table1. List of gene sets with simultaneously altered the enrichment upon SAP18 overexpression and knockdown. Gene sets enriched by SAP18 and reduced by shsap18 SAP18 against LacZ shsap18
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