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1 doi: /nture10924 no tg Lsm1-my Lsm2-my Lsm5-my Lsm6-my Lsm7-my Lsm8-my no tg Lsm1-my Lsm2-my Lsm5-my Lsm6-my Lsm7-my Lsm8-my kd SeeBlue Mrker Mgi Mrk no tg Sm1-my Smd3-my Sme1-my Smd1-my Smd2-my Smf1-my SeeBlue Mrker no tg Sm1-my Smd1-my Smd2-my Smf1-my Smd3-my Sme1-my kd Mgi Mrk k 1.5 Sm1-my Sme1-my Lsm8-my Lsm8-my kd 220 SeeBlue Mgi Mrk Smd1-my Smd2-my * Smf1-my telomeres Supplementry Figure 1. rowth phenotypes nd expression of -My-tgged Sm, Lsm nd rt1 proteins. () Effet of integrted -My epitope tgs on growth nd viility of S. pome ells. Cells of the indited strins were grown in yest extrt plus supplements 32 (YES) to lte log phse, ounted, onentrted to 1.3 x 10 8 ells ml -1 nd 3 μl of 5-fold seril dilutions were spotted to YES gr pltes followed y inution t 32 C for 3 dys. () Reltive expression level of tgged proteins. Cell extrts were prepred s desried 32 nd nlysed y western lot with nti--my ntiody. non-speifi nd reognized y nti--my in ll S. pome extrts ws used s loding ontrol (). Full length rt1 nd Smd2 protein re expressed t low levels, ut n e seen on longer exposures of western lots (lower pnel), where they re indited with dot (rt1) nd sterisk (Smd2), respetively. he SeeBlue prestined mrker ws used to onfirm trnsfer from the gel onto the memrne, ut is not visile on the film. () elomere length nlysis for strins expressing tgged rt1, Sm nd Lsm proteins. enomi DN ws digested with EoRI nd nlysed y Southern lotting with telomeri proe s desried 32. frgment of the rd16 gene ws used s loding ontrol (). 1
2 RESERCH SUPPLEMENRY INFORMION Sm1-my Sme1-my Smf1-my Smd2-my untgged * snrn U1 untgged rt1 IP Lsm4 IP Sm1 IP C Lsm6-my Lsm7-my Lsm2-my Lsm5-my < tivity Lne snrn U6 d * Sm1-my Sme1-my Lsm8-my Supplementry Figure 2. Co-immunopreipittion of with Sm nd Lsm proteins. () Northern lot for nd snrn U1 on RN isolted from nti--my immunopreipittions from strins hrouring the indited epitope-tgged proteins. he sterisk mrks the position of the preursor visile in Sm immunopreipittes. () Northern lotting nlysis s in () for dditionl tgged Lsm proteins. () elomerse ssy nd northern lot for RN reovered from IPs s indited. No tivity ws deteted in n untgged ontrol. (d) Northern lot nlysis for from the rt1, Sm nd Lsm immunopreipittion used in the telomerse ssy shown in Fig. 1d. he sterisk mrks the position of the preursor. he mount of mture reovered in eh IP is normlized to the rt1 IP smple. 2
3 RESERCH ter1-sm6 100% 100% 20% 10% 5% snrn U6 (wt) ter1-sm ter1-sm U ter1-sm U 2 ter1-sm U 3 preursor mture (wt) ter1-sm ter1-sm U ter1-sm U 2 ter1-sm U 3 R Supplementry Figure 3. Effets of muttions in the Sm site. () Loss of the Sm site ompromises Lsm ssoition. Lsm4 immunopreipittions were rried out in prllel from ell extrts ontining or the ter1-sm6 mutnt followed y northern nlysis. Wheres 5% of ws redily deteted, ter1-sm6 ws undetetle. U6 snrn served s ontrol. () Shortening the Sm site ompromises proessing. Deleted nuleotides re indited in the mutnt nmes. Northern lot for nd snorn snr101 s loding ontrol (). () Spliing of mutnts ontining prtil deletions of the Sm site ws exmined y R PCR ross the intron using primers BLoli1275 nd BLoli
4 RESERCH SUPPLEMENRY INFORMION vetor Sm1 Smd3 Smd1 Sme1 Smf1 Smg1 Smd2 vetor Lsm1 Lsm2 Lsm3 Lsm4 Lsm5 Lsm6 Lsm7 Lsm8 Smd3 ter1-sm6 100% α-m IP (wt) 20% 10% 4% -His -de snrn U1 d e R ll forms input ter1-5 ssmut-hh * IP input IP Mrker p 1, / / Lne UUUUUU gcuguuugugguguguuuugguuguggugugggggg Sm site 5 ss hmmerhed riozyme sequene input α-m IP 100% 100% 20% 10% 5% f uuuguuuuuuuuuuuuuuuuguu rnh site 3 ss 1 C.12 tivity 1 2 Lne g Input Lsm4-IP Sm1-IP rtio (/ ) Supplementry Figure 4. Chrteriztion of gs1 nd the M p on. () Yest two-hyrid ssys using gs1 s it with Sm nd Lsm proteins s indited. Columns represent 3-fold dilutions on non-seletive medium (top) nd seletive medium (ottom). () Loss of Sm site ompromises M p formtion. Northern lot of nd ter1-sm6 from α-m IP smples. dilution series for the wild type smple ws inluded to show tht 4% of levels re detetle y this method. () Preursor nd splied form re not M-pped. RN isolted from input nd α-m IP smples ws sujeted to R-PCR nlysis to detet the presene of (ll forms, lnes 1 to 4) or speifilly the preursor nd splied forms (lnes 5 to 8). R=reverse trnsriptse. n sterisk mrks non-speifi nd not reproduile nd oserved in lne 1. (d) gs1 is responsile for 5 p hypermethyltion on. Northern nlysis of from α-m IP smples from nd strins. (e) Shemti of ter1-5 ssmut-hh mutnt. he 5 splie site (5 ss) ws mutted nd hmmerhed riozyme sequene ws inserted downstrem. he hmmerhed levge site is indited with vertil rrow. (f) Deletion of does not impir telomerse tivity reovered from Lsm4 IPs eyond the effet expeted from the redued level in ells. tivity of the smple is indited reltive to. 32 P-lelled 100mer oligonuleotide ws used s reovery nd loding ontrol (). (g) Northern lot for using RN isolted from Lsm4 nd Sm1 immunopreipittions. For input nd eh IP, the rtio etween the tgs1 deletion nd is shown elow the lne. 4
5 RESERCH 3 end distriutions (%) UUUUUU -UUUUUU -UUUUU -UUUU -UUU -UU -U Sme1 Smf1 3 end distriutions (%) UUUUUU -UUUUUU -UUUUU -UUUU -UUU Lsm3 Lsm4 -UU rt1 -U 3 end distriutions (%) UUUUUU -UUUUUU -UUUUU -UUUU -UUU lsm3 (smple 1) lsm3 (smple 2) -UU -U d lsm3 Input Pellet Superntnt lsm3δ α-m IP lsm3 lsm3δ lsm3 lsm3δ Lne Supplementry Figure 5. Lsm protets the 3 end of the mture form of. () 3 end sequene nlysis of from Sme1 nd Smf1 IPs. verge nd stndrd devition from two experiments, numer of sequenes nlysed: 3.9 x 10 6 (Sme1) nd 7.9 x 10 6 (Smf1). () s () for Lsm3 (four experiments, 21.2 x 10 6 sequene reds), Lsm4 (three experiments, 11.4 x 10 6 sequene reds) nd rt1 (three experiments, 15.2 x 10 6 sequene reds). () Speifi loss of Lsm2-8 ound frtion of in lsm3δ ells sed on 3 end sequene nlysis from two totl RN smples (1.7 x 10 6 nd 3.3 x 10 6 sequene reds were nlysed). he smple from Fig.1 is inluded for omprison. (d) Deletion of lsm3 ffets proessing nd stility ut not 5 gunosine p hypermethyltion. Northern lot for with RN isolted from α-m IP smples from nd lsm3δ strins. 5
6 RESERCH SUPPLEMENRY INFORMION otl RN SmB1 IP Lsm4 IP rt1 IP UUUU UUUU UUU UUU UU U 0% 20% 40% 60% 80% 100% Supplementry Figure 6. 3 end sequene distriution for ter1-smu1 ws determined y mssively prllel sequening from totl RN nd nti--my immunopreipittes from strins hrouring -My tgged Sm1, Lsm4 or rt1, respetively. Eh r shows the frtions of end sequenes in different olours. Note tht the intt SmU1 site (UUUU) represents over 60% of the totl s ompred to 35% for the (Fig 1) onsistent with diminished Lsm inding leding to preferentil degrdtion of shorter forms. he intt SmU1 site is predominntly reovered with Sm1, wheres Lsm4 nd rt1 strongly enrih the trunted forms. otl numer of sequene reds from two experiments used for the nlysis were s follows: 5.6 x 10 6 (totl RN), 6.0 x 10 6 (Sm1), 5.0 x 10 6 (Lsm4), nd 2.7 x 10 6 (rt1). 6
7 RESERCH le S1. Yest Strins Used in his Study Strin Numer enotype Soure PP138 h - de6-m216 leu1-32 ur4-d18 his3-d1 L stok PP298 h - de6-m210 leu1-32 ur4-d18 his3-d1 trt1-my 9 Ref. 33 PP399 leu1-32 ur4-d18 his3-d1 trt1-cmy 9 ter1:: knmx6 L stok PP407 h /h - leu1-32/leu1-32 ur4-d18/ur4-d18 his3-d1/ his3-d1 Ref. 2 de6-m210/de6-m216 ter1 / ter1::knmx6 PP433 h /h - leu1-32/leu1-32 ur4-d18/ur4-d18 his3-d1/ his3-d1 de6-m210/de6-m216 ter1 / ter1::ur4 his study PP574 h - de6-m216 leu1-32 ur4-d18 his3-d1 lsm2-my 13 -nt his study PP575 h - de6-m216 leu1-32 ur4-d18 his3-d1 lsm1-my 13 -nt his study PP576 h - de6-m216 leu1-32 ur4-d18 his3-d1 lsm3-my 13 -nt his study PP577 h - de6-m216 leu1-32 ur4-d18 his3-d1 lsm4-my 13 -nt his study PP578 h - de6-m216 leu1-32 ur4-d18 his3-d1 lsm5-my 13 -nt his study PP579 h - de6-m216 leu1-32 ur4-d18 his3-d1 lsm6-my 13 -nt his study PP580 h - de6-m216 leu1-32 ur4-d18 his3-d1 sm1-my 13 -nt his study PP582 h - de6-m216 leu1-32 ur4-d18 his3-d1 sme1-my 13 -nt his study PP583 h - de6-m216 leu1-32 ur4-d18 his3-d1 lsm7-my 13 -nt his study PP584 h - de6-m216 leu1-32 ur4-d18 his3-d1 smd2-my 13 -nt his study PP585 h - de6-m216 leu1-32 ur4-d18 his3-d1 lsm8-my 13 -nt his study PP588 h - de6-m216 leu1-32 ur4-d18 his3-d1 smf1-my 13 -nt his study PP670 h de6-m210 ur4-d18 his3-d1 tgs1::knmx6 his study, sed on Ref. 22 PP677 h de6-m216 ur4-d18 leu1-32 lsm1::knmx6 his study, sed on diploid strin from Bioneer PP678 h de6-m216 ur4-d18 leu1-32 lsm3::knmx6 his study, sed on diploid strin from Bioneer PP694 de6-m216 leu1-32 ur4-d18 his3-d1 ter1::knmx6 lsm4- his study my 13 -nt PP695 de6-m216 leu1-32 ur4-d18 his3-d1 ter1::knmx6 sm1- his study my 13 -nt PP758 leu1-32 ur4-d18 his3-d1 ter1::knmx6 lsm4-my 13 -nt his study PP759 leu1-32 ur4-d18 his3-d1 ter1::knmx6 sm1-my 13 -nt his study 7
Supplementary Figure S1. Akaike et al.
reltive expression of HIPK2 (HIPK2/GAPDH) Supplementry Figure S1. Akike et l. 1.25 ontrol HIPK2 #1 1 HIPK2 #2 HIPK2 3 U 0.75 0.5 0.25 0 ontrol : + + - HIPK2 #1: - - - + + - - - - HIPK2 #2: - - - - + +
Gan et al., Supplemental Figure 1
Gn et l., Supplementl Figure IB: DU45 Unp IB: py-68 IB: perk IB: ERK IB: Akt Unp DU45 DU45 ErB2 - EGF - EGF ErB2 75 75 Supplementl Figure. DU45 nd ells predominntly express nd re highly responsive to EGF.
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DOI: 1.138/n74 In the formt provided y the uthors nd unedited. d 331p 637p 9p 394p 48p 467p 22p 23p 489p 419p 3p 493p 332p 53p 39p 1 G1: 16.4% S: 73.1% G2/M: 1.5% 2 1 2 E-KO G1: 2.2% S: 7.7% G2/M: 9.1%
sensitive VBSs Vh subdomains EF EF
Tlin- Tlin-EGFP-His 2 3 2 3 ABD Mehno- ABD2 ABD3 sensitive VBSs DD 6xHis EGFP Vh sudomins EGFP-Vinulin EGFP 2 3 4 Vt EGFP-Vh EGFP 2 3 4 -tinin- CH CH2 SP SP SP SP EF EF mcherry--tinin- mcherry CH CH2 SP
HeLa. CaSki. Supplementary Figure 1. Validation of the NKX6.1 expression in mrna level and
Supplementry Figure. Li et l. Reltive expression ( / GAPDH ) 6 5 4 3 2 5 Reltive expression ( / GAPDH ) 4 3 2 Reltive expression ( / GAPDH ) 4 3 2 Reltive expression ( / GAPDH ) 4 3 2 Ve S Ve S2 S S2 Ve
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Pulldown HeL lyste lyste lyste 25 5 75 5 5kD 37 25 5 MDDIFTQCREGNAVAVRLWLDNTENDLNQGDDHGFSPLHWACREGRSAVVEMLIMRGARINVMNRGDDTP LHLAASHGHRDIVQKLLQYKADINAVNEHGNVPLHYACFWGQDQVAEDLVANGALVSICNKYGEMPVDKA KAPLRELLRERAEKMGQNLNRIPYKDTFWKGTTRTRPRNGTLNKHSGIDFKQLNFLTKLNENHSGELWKG
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1 SUPPLEMENTRY FIGURES Fig. 1 & Specificity of the nti-p-s211 nd nti-p-s226 ntibodies COS-1 cells trnsiently trnsfected with either H hgr wt, H hgr S211 or H hgr S226 were treted with 1nM Dex for 1 hour.
Supplemental Figure S1
Supplementl Figure S1 TG nrt1.5- Li et l., 1 nrt1.5- Lin et l., 8 F L CTGCCT R T 5'UTR 3'UTR 1 3 81p (k) nrt1.5- C nrt1.5- Supplementl Figure S1. Phenotypes of the T-DN insertion mutnts (this pper), nrt1.5-
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S shrna S Viility, % of NT sirna trnsfete ells 1 1 Srmle NT sirna Csp-8 sirna RIP1 Atin L929 shrna sirna: Csp8 Atin L929: shrna Csp8 e Viility, % of NT sirna trnsfete ells NT sirna Csp-8 sirna M45 M45mutRHIM
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Supplementry Figure S1. depletion indues poptosis vi the spse-8 pthwy. Atin 61.5 46.2 48h 72h 96h zvad 96h 5. % 6.8 % 8.7 % 4.3% simk1#5 9.5 % 22.5 % 39.6 % 11.8 % simk1#6 8. % 21.7 % 33.5 % 11. % 48h
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2000 Nature America Inc.
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