doi:.8/nture98 : hr NEMO :5 hr IKK IKK NF-κB p65 p5 p65/-rel NF-κB p65 p5 p65/-rel Cytoplsm Cytoplsm p65/p5 Nuleus Nuleus NEMO IKK IKK d : hr > : hr p65/-rel NF- p65 p5 Cytoplsm Cytoplsm p65/p5 p65/-rel p65/p5 p65/-rel Nuleus Nuleus Supplementry Figure exerts oth typil nd typil effets on NFκB regultion of TNFα expression.,, like funtions s typil IκB fmily memer y sequestering NF κb dimers in the ytoplsm. seletively inds p65 or Rel ontining omplexes through diret inding to either p65 or Rel. lso inds nd stilizes the p65: Rel heterodimer whih is otherwise not present., LS stimultion through TLR4 nd multiple intervening signling intermedites tivtes the IKK omplex resulting in phosphoryltion of nd its susequent uiquitintion nd degrdtion. Relesed dimers trnslote to the nuleus where they ind κb sites in the promoters/enhners of vrious genes inluding TNFα, nd Rel., phosphoryltion nd degrdtion ours with slower kineti resulting in the relese of NF κb inluding p65: Rel heterodimers whih ind to seletive κb sites inluding speifi κb site (κb) in the TNFα promoter. d, Newly synthesized inds nd sequesters NF κb omplexes in the ytoplsm terminting trnsription of most NF κb dependent genes. Newly synthesized hypophosphorylted inds to p65: Rel forming trimeri omplex tht is resistnt to inding nd is thus ple of ontinued intertion with the κb site of the TNFα promoter nd responsile for ontinued expression of TNFα.
doi:.8/nture98 proe X R Wild-type llele Trgeting vetor Trgeted llele 9.k 5.7k tk X R 9. k R RXneor H R 5.7 k X RXneo r H d +/+ +/+ +/- +/- wt ko +/- +/+ -/- H +/+ +/- -/- Supplementry Figure Genertion of the / mie, The struture of the gene, the trgeting vetor nd the predited disrupted gene. Blk oxes denote the exons. Restrition enzymes: X, XI; R, EoR; H, HindIII. : primers used for CR genotyping of WT llele ( nd ) nd trgeted llele ( nd )., Sreening for homologous reomintion etween the trgeting vetor nd the endogenous gene. ES ell genomi DNA ws digested with XI, eletrophoresed nd hyridized with rdioleled proe indited in A., CR genotyping of offspring from the heterozygote interrosses. Til DNA ws otined nd CR with primers indited in A ws rried out. d, / mie do not express. Western lot nlysis of totl ell lyste of MEF ells derived from.5 dy emryos.
doi:.8/nture98 IκΒβ IκΒα IκΒε GADH Thymus Hert Lung Liver Spleen Kidney Intestine Lymph Node IκΒβ IκΒα IκΒε 65 RelB -Rel 5 GADH wt +/- -/- NF-κB tivity (K) 6 8 8.9 4. IκΒβ +/+ IκΒβ -/- d NF-κB tivity (RLU) (K) pdna - TNF + TNF IκΒβ IκΒα Supplementry figure NF κb is tivted normlly in / MEFs., ug of totl ell lyste from vrious mouse tissues ws eletrophoresed nd nlyzed y Western lot with the indited ntiodies., ug of totl ell lyste from MEFs ws nlyzed y Western lot with indited ntiodies., Bsl NF κb DNA inding tivity ws nlyzed y EMSA with nuler extrts prepred from MEFs. The intensity of the gel shift nd ws mesured y densitometry, normlized to tht of NF Y nd grphed. d, NF κb luiferse ssy. 9 ells were trnsiently trnsfeted with pbiix lu together with Renill luiferse vetor nd pdna vetors tht express either or. Twenty four hours fter trnsfetion, ells were stimulted with TNFα (ng/ml) for 4 hours, lysed nd ssyed for luiferse tivity. Results re expressed s reltive luiferse units (RLU) normlized y Renill luiferse tivity.
doi:.8/nture98 LS: NF-κB NF-Y IκBε GADH WT.5.5.5.5 4 /.5.5.5.5 4 hrs NF-kB tivity Reltive inding 6 4 Wt / 4 Hours IL-β: NF-κB NF-Y IκBε GADH WT.5.5.5.5.5 /.5.5.5.5.5 hrs Reltive NF-kB inding tivity 8 6 4 Wt / 4 Hours WT / TNFα: NF-κB NF-Y IκBε GADH.5.5.5.5.5.5.5.5.5.5 Reltive NF-kB inding tivity Supplementry Figure 4 NF κb is normlly tivted in / MEFs. hrs 5 5 4 Hours Cells were treted with LS (ug/ml; ), IL β (ng/ml; ) or TNF α (ng/ml; ) for the indited times, then ytosoli nd nuler extrts were prepred. Nuler extrts were nlyzed for NF κb DNA inding tivity y EMSA. EMSA with NF Y DNA proe ws performed s loding ontrol. (left pnel). The intensity of the gelshift nds were quntitted y densitometry, normlized to tht of NF Y, nd grphed (right penl). Cytosoli extrts were nlyzed for, nd IκBε degrdtion y immunolotting with orresponding ntiodies. Western lot with nti GADH ntiody ws performed s loding ontrol. Wt / 4
doi:.8/nture98 F4/8 IκΒβ +/+ IκΒβ -/-.68 7.7.4 77.5..5 9.48 IκΒβ+/+ CD IκΒβ-/- TNFα mrna (% of t ) 8 6 4 5 45 Time (min) Supplementry Figure 5 Norml phenotype of nd TNFα mrna stility in thioglyolte eliited mrophges (TEM) from / mie., Chrteriztion of TEMs. 6 to 8 week old littermte mie were injeted intrperitonelly with ml of % thioglyollte roth nd three dys lter, the mie were srified nd their peritonel vities were wshed with ml old BS. Cell pellets were wshed one with DMEM supplemented with % FBS nd ultured t the onentrtion of 5X 5 ells/ml. hours lter the dishes were wshed with medium to remove nondherent ells. TEMs were stined for CD nd F4/8 nd nlyzed y flow ytometry., WT nd / TEMs generted s in () were stimulted with LS for hours, fter whih the trnsriptionl inhiitor DRB ws dded (t). The degrdtion urves represent RNA level mesured y qrt CR normlized to β tin mrna level nd expressed s perentge of the level t t. 5
doi:.8/nture98 LS 4 8 IκΒβ IκΒα hr hyperhypo- d untreted LS p65 β tulin osition Reltive to Trnsription Strt TNF-κΒ GGGG AA TCCTT -86 TNF-κΒ CGTG AA TTCCC -66 TNF-κΒ TGGGGCTGCCC -6 TNF-κΒ GGGGGCT TTCC -5 -Rel κβ onsensus GGGRNWYYCC Gene/osition Reltive to Trnsription Strt CD4-56 CD4-59 p65/rel onsensus GTAGGAATTTCCTTC TGGGGAAATCCCTGC IL -7 CTAGGAACTTCCCAG CXCL -757 CGTGGACTTTCCCTG CXCL -7 CCGGGAATTTCCCTG CXCL -669 TCGGGAAGTTCCCAA TNFα -66 TCCGTGAATTCCCAG GGAAWTTCC CD4-56 CD4-59 Cxl -7 Cxl -669 Cxl -757 Supplementry Figure 6 Diversity in NF κb inding sites in the TNFα promoter, Two phosphoryltion forms of. Rw64.7 ells were treted with LS ( mg/ml) for the indited times. Totl ell lystes were prepred nd sujeted to immunolotting with the indited ntiodies., The four NF κb sites on TNFα promoter. R, N, W nd Y denote purine, ny nuleotide, A or T, nd pyrimidine ses., otentil p65: Rel inding NF κb sites on the promoter of genes whose expression pttern is similr to TNFα in mirorry nlysis. Sites were hosen sed on the following riteri: fit 9p κb onsensus site preferred y p65 nd Rel; extr G t 5 end of the onsensus sequene is less preferred; five or six A/T sepirs t the enter of inding site is preferred (Hung, D. B., helps CB, Fuso AJ, Ghosh G. JOURNAL OF MOLECULAR BIOLOGY, 46, p47 6, 5) d, Rw 64.7 ells were left unstimulted (open rs) or were stimulted with ng/ml LS for hours (losed rs), nd were then fixed with % formldehyde for 5 min t room temperture, wshed twie with ie old BS, nd re suspended in ChI lysis uffer. The lystes were susequently sonited to sher the genomi DNA into frgments of pproximtely p. ChI nlysis ws performed on the superntnts with nti, nti Rel nd nti p65 ntiodies, nd preipitted DNA were purified nd sujeted to qcr with primers flnking eh of the indited sites of respetive promoters. 6
doi:.8/nture98..5 R.L.U...5. +/+ wt - - - - + + + + +/+ κβ -/- wt -/- κβ +/+ wt +/+ κβ -/- wt -/- κβ LS Supplementry Figure 7 TNFα κb is required for optiml tivtion of TNFα trnsription in +/+ ut not in / mrophges. +/+ nd / one mrrow derived mrophges were trnsfeted with mouse TNFα luiferse reporters s indited nd phrl TK (romeg) s n internl ontrol. 4 hours lter, the trnsfeted ells were stimulted with LS for 4 hours nd luiferse tivity were plotted s reporter (firefly)/ontrol (renill). 7
doi:.8/nture98 IκΒβ +/+ IκΒβ -/- Reltive TNFα mrna level 5 Time (hours) Reltive IL-6 mrna level..8.4 5 Time (hrs) Reltive IL-β mrna level 5 Time (hours) Supplementry Figure 8 Reltive mrna expression level of TNFα, IL 6 nd IL β gene. BMDM from oth wild type ( +/+ ) nd / were stimulted with LS (mg/ml) for the indited times. Reltive mrna undne ws determined y qrt CR (normlized using β tin mrna levels). 8
doi:.8/nture98 Averge S ore +/-S E M 7.5 5..5 Clinil S ore. KO Wt. 8 5 4 Averge thikness +/-S E M pd thikness KO Wt 9 8 7 6 5 8 5 4 d Supplementry Figure 9 knok out mie exhiited delyed onset nd ttenuted one destrution in n experimentl model of CIA. Severity of rthritis, ssessed y linil soring () or pw thikness mesurement (), in wild type DBA or knok out mie (krossed for eight genertions to the DBA kground)., Representtive exmples of the pw swelling in mie from wild type DBA (left) or knok out (right) mie t dy 8. d, Representtive histologil nlysis of H&E stined (upper) setions of joints from wild type DBA or knok out mie. Left pnel, lrgely intt joint of knok out mouse (Clinil sore ). Middle pnel, joint of knok out mouse (Clinil sore ), exhiited serious synovil tissue prolifertion nd inflmmtory infiltrtion, however, one nd rtilge destrution ws miniml. Right pnel: joint of wild type DBA mouse (Clinil sore 4), exhiited extensive inflmmtory ell infiltrtion nd severe destrution of one/joint struture. 9
doi:.8/nture98 45 4 5 5 5 5 MI-α RANTES MC Supplementry Figure knok out mie exhiited norml prodution of MI α, RANTES nd MC in n experimentl model of CIA. Serum MI α (), RANTES (), MC () levels in wild type DBA (filled rs) or knok out (open rs) mie.