plasmids and constructs  human socs-3 cdna was amplified from 
  a human testis cdna library ( bd biosciences ) by pcr using specific primers 
  ( forward , 5-atggtcacccacagcaagtttccc-3 ; reverse , 
  5-ttaaagcggggcatcgtactggtc-3 ) and pyrobest dna polymerase 
  ( takara ) .
the pcr conditions were as follow : preincubation at 98 c for 20 
  s and then 28 cycles of denaturation at 98 c for 15 s , annealing at 70 
  c for 20 s , and extension at 72 c for 2 min 30 s. the pcr products 
  were electrophoresed on 1.5% agarose gels , and fragments were extracted using 
  a qiaex ii kit ( qiagen ) .
these extracted fragments were amplified by pcr under 
  the same conditions using specific primers containing ecori and bamhi sites . 
 
the pcr products were digested with ecori and bamhi and cloned into the same 
  sites of pgbt9 ( bd biosciences ) . for generation of flag - socs-3 ( wt ) , socs-3 
  cdna
was digested by ecori and sali from socs-3pgbt9 and cloned into the ecori 
  and xhoi sites of flag - pcdna3 ( invitrogen ) .
cdna fragments of socs-3 deletions 
  were amplified from full - length socs-3 cdna by pcr using specific primers and 
  pyrobest dna polymerase ( takara ) .
gfp - dp-1 and 6xmyc - dp-1 expression plasmids 
  were previously described 
  ( 27 ) .
hdp-2 cdna also was 
  amplified from a human testis cdna library ( bd biosciences ) by pcr using 
  specific primers ( forward , 
  5-atgattataagcacaccacagagactaaccagttcagg-3 ; reverse , 
  5-gaaacgtaggctttctcttgtctttattctggggag-3 ) .
hdp-2 and hdp-3 cdnas were amplified by pcr using 
  specific primers containing ecori and xhoi sites .
yeast cultures , the preparation of 
  yeast selection media , and yeast transformations were carried out according to 
  the manufacturer 's protocol ( bd biosciences ) . in the first step of the 
  two - hybrid library screening procedure , 100 ng of pgbt9-hsocs-3 plasmid ( bait ) 
 
was used to transform ah109 yeast strain containing 4 reporter genes , i.e. 
  lacz , mel1 , his3 , and ade2 .
the transformants were mated with a 
  pretransformed human skeletal muscle cdna library in yeast strain y190 ( bd 
  biosciences ) . in the second step ,
the transformants were selected for growth 
  on media lacking tryptophan , leucine , histidine , and adenine and containing 
  glucose as the carbon source .
interactors were selected by plating on the same 
  medium containing the substrate of -galactosidase .
plasmids were extracted from leu+ colonies and 
  used to transform escherichia coli ( dh5 ) .
cell culture and transfection of hek 293 cells  human 
  embryonic kidney ( hek ) 293 cells were cultured at 37 c with 5% 
  co2 in dulbecco 's modified eagle 's medium ( dmem ; sigma ) containing 
  100 units / ml penicillin , 100 g / ml streptomycin , and 10% fetal calf serum 
  ( thermo trace ) as previously described 
  ( 27 ) . for the luciferase and 
  cell growth assays , plasmids and sirnas were introduced into hek 293 cells 
  using lipofectamine 2000 ( invitrogen ) . in other experiments , polyfect 
  transfection reagent ( qiagen ) was also used for plasmid transfection . for 
  establishing hek 293 cells stably expressing flag - socs-3 ,
the cells were 
  cultured at 90% confluence in 6-well dishes and then transfected with 4 g 
  of plasmid expressing flag - socs-3 .
one day later , the transfected cells were 
  subcultured and grown in the presence of 1 mg / ml of g418 ( invitrogen ) . after 2 
  weeks , single g418-resistant colonies
these colonies were then maintained in medium containing 1 
  mg / ml of g418 and analyzed individually for expression of flag - socs3 .
immunoprecipitation and immunoblotting assays  hek 293 cells 
  ( 1  10 ) were transfected with flag - socs-3 and 6xmyc - dp-1 
  expression plasmids using polyfect ( qiagen ) and then cultured for 24 h. the 
  transfected cells were lysed in tne buffer ( 10 mm tris - hcl , ph 7.9 , 
  150 mm nacl , 1 mm edta ) containing 0.1% nonidet p-40 and 
  a protease inhibitor mixture ( roche applied science ) .
c57bl/6jjcl mouse 
  ( 1214 weeks old)-derived lung and testis were homogenized in tne buffer 
  using a pellet pestle ( kontes ) .
after centrifugation at 13,500 rpm for 10 min 
  at 4 c , the supernatants ( 5 mg of total proteins ) were used for each 
  immunoprecipitation ( ip ) assay .
proteins in the cell lysate or tissue extracts 
  were precipitated for 1 h at 4 c with anti - flag m2-agarose ( sigma ) or 
  anti - socs-3 polyclonal antibody ( ibl ) bound to protein a - sepharose ( sigma ) . 
 
the immunocomplexes were washed five times with tne buffer . after having been 
  washed ,
the antigen of the immunocomplexes obtained with anti - flag m2-agarose 
  was eluted using flag peptide ( sigma ) , and the supernatant was suspended in 
  2 sds - page sample buffer and boiled for 2 min at 98 c .
the 
  immunocomplexes formed with anti - socs-3 polyclonal antibody bound to protein 
  a - sepharose were directly suspended in 2 sds - page sample buffer and 
  boiled .
the samples were separated by sds - page and analyzed by immunoblotting 
  using mouse anti - flag m2 monoclonal antibody ( sigma , 1:1,000 dilution ) , mouse 
  anti - myc monoclonal antibody 9e10 ( santa cruz biotechnology , 1:1,000 
  dilution ) , anti - socs-3 antibody or anti - dp-1 monoclonal antibody ( genetex , 
  1:1,000 dilution ) as first antibodies .
after four washes with tbst , incubation 
  with secondary antibody was performed using peroxidase - conjugated goat 
  anti - rabbit igg ( for anti - socs-3 , abcam , 1:2,000 dilution ) or 
  peroxidase - conjugated rabbit anti - mouse igg ( for other monoclonal antibodies , 
  dako japan , 1:1,000 dilution ) . finally , after four more washes with tbst , the 
  immunoblots were apposed to hyperfilm ( ge healthcare ) , which was then exposed 
  for 15 s to 2 min
hek 293 
  cells were divided into several 35-mm glass bottom culture dishes ( mattec 
  corp . ) and transfected with the gfp - empty , gfp - dp-1 , and/or flag - socs-3 
  plasmids .
transfected cells were grown for 24 h at 37 c in dmem 
  containing 10% fetal bovine serum .
the cells were then washed three times with 
  pbs ( - ) and fixed in 4% paraformaldehyde / pbs for 30 min at room temperature . 
 
they were then again washed three times with pbs ( - ) . for the detection of 
  flag - socs-3 ,
after the wash with pbs ( - ) , the cells were incubated with 
  anti - flag m2 antibody ( sigma ) in blocking solution at 1:1000 dilution for 1 h. 
  they were subsequently washed five times with 0.1% triton x-100/pbs ( pbst ) and 
  thereafter incubated with cy-5-labeled anti - mouse igg ( chemicon international ) 
  in blocking solution at 1:1000 dilution for 1 h. finally , the cells were 
  washed five times with pbst , and the coverslip was mounted with vectashield 
  ( vector lab , inc . ) .
samples were examined using an lsm 510 meta confocal 
  imaging system ( carl zeiss ) .
hek 293 cells stably expressed flag - socs-3 were 
  also cultured in 35-mm glass bottom dishes and fixed by the same methods .
the 
  fixed cells were then incubated with anti - socs-3 polyclonal antibody ( ibl ) and 
  anti - dp-1 monoclonal antibody spm178 ( genetex ) in blocking solution at 1:1000 
  dilution for 1 h. they were subsequently washed five times with pbst and 
  thereafter incubated with cy-5-labeled anti - mouse igg ( chemicon international , 
  1:1000 dilution ) and alexa fluor 488-labeled anti - rabbit igg ( molecular 
  probes , 1:2000 dilution ) in blocking solution for 1 h. the samples were then 
  examined using an fv-1000 confocal imaging system ( olympus ) .
hek 293 cells were cultured in 24-well 
  microplates and transfected with 100 ng of cyclin - e - luc or apre - luc reporter 
  plasmids , 10 ng of prl - tk - luc internal control plasmid , and the desired 
  expression plasmids ( 100 ng of each expression plasmid ) .
the total amount of 
  plasmid dna was kept constant by balancing with empty expression plasmids . 
 
transfected cells were cultured for 24 h. for the apre - luc assay , cells were 
  incubated for 6 h with or without 10
luciferase activity was detected with a veritas microplate luminometer 
  ( promega ) and dual - luciferase reporter assay system ( promega ) .
chromatin immunoprecipitation ( chip)hek 293 cells were 
  cultured in 12-well microplates and transfected with expression plasmids and 
  sirna .
cells were fixed by directly adding formaldehyde to the medium ( final 
  concentration 1% ) .
15 min later , the fixed cells were washed with ice - cold pbs 
  containing protease inhibitors .
the cells were solubilized in lysis buffer , 
  and then chromatin was sheared to an average dna fragment size of 
  200500 bp by sonication .
chip assays were performed using one day chip 
  assay kit ( diagenode ) according to the manufacturer 's protocol .
antibodies 
  used for immunoprecipitation included anti - dp-1 monoclonal antibody spm178 
  ( genetex ) and anti - e2f monoclonal antibody ( upstate ) . with the use of prime 
  star ( takara )
, the eluted dna was amplified by pcr with the following primers 
  for the cyclin - e promoter ( encompassing the 3 major e2f binding sites ; 
  forward , 5-cgcccgccgtgtttacattccac-3 ; reverse , 
  5-cgaggcgcagggacggggaatc-3 ) .
hek 293 cells were cultured in 10-cm dishes 
  and transfected with the desired plasmids .
24 h later , the cells were 
  harvested and then stained with cycletest plus ( bd biosciences ) .
subsequently , 
  flow cytometric analysis was carried out in a facscalibur flow cytometer ( bd 
  biosciences ) using cellquest software .
were transfected 
  with the indicated expression plasmids , and then cultured for 2 days in 10% 
  fetal bovine serum containing dmem .
cell growth was examined by performing a 
  celltiter - glo luminescent cell viability assay ( promega ) .
short - interfering rna ( sirna ) transfection  the following 
  sirna oligonucleotides were used to interrupt the expression of endogenous and 
  exogenous socs-3 or dp-1 : socs3 stealth select rnai ( catalog number , 
  hss113313 , invitrogen ) , dp-1 stealth select rnai ( catalog number , hss144254 , 
  invitrogen ) , and stealth rnai negative control med gc duplex 3 ( catalog 
  number , 12935 - 113 , invitrogen ) .
hek 293 cells were transfected with these 
  sirnas according to the manufacturer 's protocol ( invitrogen ) with the aid of 
  lipofectamine 2000 ( invitrogen ) .
identification of dp-1 as a socs-3-interacting protein  using 
  yeast two - hybrid screening and a human testis cdna library , we sought proteins 
  that could interact with socs-3 .
we initially found that 28 of 76 clones were 
  the c - terminal portion of the transcription factor dp-1 . by this screening
, 
  four types of dp-1 clones ( 103410 , 192410 , 238410 , 
  240410 amino acids ) were identified .
these results suggest that the 
  c - terminal domain of dp-1 ( 240410 amino acids ) was the region 
  interacting with socs-3 . socs-3
interacts with dp-1 in cells in vitro and in vivo  the 
  interaction between socs-3 and dp-1 in hek 293 cells was investigated by 
  conducting an immunoprecipitation - western blot assay .
the cell lysates were 
  examined for the expression of flag - socs-3 and 6 myc - dp-1 proteins 
  ( fig .
the lysates were first immunoprecipitated with flag antibody 
  beads , which were then washed and examined by western blotting assay with a 
  flag or myc antibody . as shown in fig . 
  1 , socs-3 bound dp-1 ( fig . 
 
in addition , dp-1 also interacted 
  with socs-1 and socs-2 ( data not shown ) .
1a ) , we 
  determined the critical region in socs-3 required for interaction with dp-1 . 
 
socs-3 having an n- or c - terminal truncation , c188 and n45 , were 
  able to bind dp-1 ( fig . 
 
1ba , lanes 3 and 4 ) , but c141 
  could not ( fig . 
 
therefore , we created a further 
  deletion between 141 and 188 and examined the ability of this mutant to bind 
  dp-1 .
in addition , although we prepared c - terminally truncated forms of 
  dp-1 to address the domain interacting with socs-3 , these proteins were 
  expressed at low levels , making such an assessment impossible ( data not 
  shown ) .
b , interaction of flag - socs-3 with 6xmyc - dp-1 was 
  analyzed using the immunoprecipitation - western blotting assay .
hek 293 cells 
  were transiently transfected with plasmids expressing either flag - socs-3 or 
  6myc - dp-1 .
the immunoprecipitates ( a and b ) or cell 
  lysates ( c and d ) were blotted with anti - flag ( b 
  and c ) or anti - myc ( a and d ) antibodies . 
 
c , interaction of socs-3 with endogenous dp-1 in cells in 
  vitro and in vivo was analyzed using the 
  immunoprecipitation - western blotting assay .
lysates of hek 293 cells ( lane 
  1 ) , hek 293 cells stably expressing flag - socs-3 ( lane 2 ) and 
  extracts prepared in tne buffer by homogenization of lung and testis excised 
  from a normal c57bl/6jjcl male mouse ( 1214 weeks old , lanes 3 
  and 4 ) were used for immunoprecipitation - western blotting . 
 
d , interaction of flag - socs-3 with 6xmyc - dp-1 ( wt , lane 1 ) , 
  dp-1 (  , lane 2 ) , dp-1 (  , lane 3 ) , dp-2 ( wt , 
  lane 4 ) , and dp-3 ( wt , lane 5 ) , was analyzed using the 
  immunoprecipitation - western blotting assay .
b , interaction of flag - socs-3 with 6xmyc - dp-1 was 
  analyzed using the immunoprecipitation - western blotting assay .
hek 293 cells 
  were transiently transfected with plasmids expressing either flag - socs-3 or 
  6myc - dp-1 .
the immunoprecipitates ( a and b ) or cell 
  lysates ( c and d ) were blotted with anti - flag ( b 
  and c ) or anti - myc ( a and d ) antibodies . 
 
c , interaction of socs-3 with endogenous dp-1 in cells in 
  vitro and in vivo was analyzed using the 
  immunoprecipitation - western blotting assay .
lysates of hek 293 cells ( lane 
  1 ) , hek 293 cells stably expressing flag - socs-3 ( lane 2 ) and 
  extracts prepared in tne buffer by homogenization of lung and testis excised 
  from a normal c57bl/6jjcl male mouse ( 1214 weeks old , lanes 3 
  and 4 ) were used for immunoprecipitation - western blotting . 
 
d , interaction of flag - socs-3 with 6xmyc - dp-1 ( wt , lane 1 ) , 
  dp-1 (  , lane 2 ) , dp-1 (  , lane 3 ) , dp-2 ( wt , 
  lane 4 ) , and dp-3 ( wt , lane 5 ) , was analyzed using the 
  immunoprecipitation - western blotting assay .
next , to confirm this interaction in cells in vitro as well as 
  in vivo , we used hek 293 cells stably expressing flag - socs-3 and 
  mouse lung or testis .
as shown in fig . 
  1c , we observed the interactions between socs-3 and dp-1 
  in a stable - expressed hek 293 transfectant of flag - socs-3 as well as in mouse 
  lung and testis extracts ( fig . 
 
furthermore , it was of interest to us to examine whether socs-3 also would 
  be able to interact with other dp molecules ( dp-1  ,  , dp-2 , and 
  dp-3 ) . therefore , we investigated this point using the immunoprecipitation 
  assay .
1d , we observed that dp-3 also was able to interact with 
  socs3 ( fig .
1da , 
  lane 5 ) but that dp-1  ,  , and dp-2 was not 
  ( fig .
these results strongly suggest that socs-3 is able to 
  interact with dp-1 in cells in vitro and in vivo and that 
  the 156172 region of socs-3 and the c terminus of dp-1 are required for 
  this interaction .
co - localization of socs-3 and dp-1although we found that 
  socs-3 interacted with dp-1 in hek 293 cells , it is important to observe 
  visually this cellular interaction .
therefore , we used confocal microscopy to 
  examine the localization of dp-1 and socs-3 and their interaction . hek 293 
  cells were transiently transfected with green fluorescent protein ( gfp)-fused 
  dp-1 or flag - tagged socs-3 expression plasmid or both .
the localization of 
  each protein was observed at 18 h after the transfection . when these 
  expression plasmids were used separately , although gfp was expressed 
  throughout the cells ( fig . 
 
2a ) , gfp - dp-1 was predominantly located in the cytoplasm 
  ( fig .
several 
  studies ( 28 , 
  33 , 
  34 ) also have demonstrated 
  that dp-1 overexpressed in hek 293 cells in the cytoplasm . on the other hand , 
 
2c ) . in 
  addition , we observed that both proteins were co - localized in the cytoplasm 
  after co - transfection ( fig . 
 
2 g , yellow to orange fluorescence ) ; 
  thus the expression pattern of these proteins remained unaltered 
  ( fig .
2 , d  g ) . 
  next , to confirm this co - localization we used hek 293 cells stably expressing 
  flag - socs-3 . as shown in fig .
k , although endogenously expressed dp-1 was 
  mainly expressed in the nucleus ( fig . 
 
2h ) , the co - localization was observed in the cytoplasm 
  ( fig .
these 
  results suggest that the interacting event between socs-3 and dp-1 occurred in 
  the cytoplasm of hek 293 cells .
hek 293 cells were transiently transfected with pegfp - c2 empty plasmid 
  ( a ) pegfp - c2-dp-1 ( b ) , or pcdna3-flag - socs-3 ( c ) . 
 
hek 293 cells were also co - transfected with pegfp - c2-dp-1 and 
  pcdna3-flag - socs-3 ( d  g ) .
dp-1 was detected by gfp 
  fluorescence ( d ) , whereas socs-3 was detected with anti - flag antibody 
  and cy5-conjugated secondary antibody ( e ) .
dic image ( f ) , 
  and merged gfp / cy5 fluorescence ( g ) are also shown .
hek 293 cells 
  stably expressing flag - socs-3 were also examined ( h  k ) .
dp-1 
  was detected with anti - dp-1 monoclonal antibody and cy5-labeled secondary 
  antibody ( h ) , whereas socs-3 was detected with anti - socs-3 polyclonal 
  antibody and alexa fluor 488-labeled anti - rabbit igg ( i ) .
the dic 
  image ( j ) and merged alexa fluor 488/cy5 fluorescence ( k ) 
  are also shown .
yellow - to - orange staining indicates co - localized 
  socs-3 and dp-1 ( g and k ) .
hek 293 cells were transiently transfected with pegfp - c2 empty plasmid 
  ( a ) pegfp - c2-dp-1 ( b ) , or pcdna3-flag - socs-3 ( c ) . 
 
hek 293 cells were also co - transfected with pegfp - c2-dp-1 and 
  pcdna3-flag - socs-3 ( d  g ) .
dp-1 was detected by gfp 
  fluorescence ( d ) , whereas socs-3 was detected with anti - flag antibody 
  and cy5-conjugated secondary antibody ( e ) .
dic image ( f ) , 
  and merged gfp / cy5 fluorescence ( g ) are also shown .
hek 293 cells 
  stably expressing flag - socs-3 were also examined ( h  k ) . dp-1 
  was detected with anti - dp-1 monoclonal antibody and cy5-labeled secondary 
  antibody ( h ) , whereas socs-3 was detected with anti - socs-3 polyclonal 
  antibody and alexa fluor 488-labeled anti - rabbit igg ( i ) . the dic 
  image ( j ) and merged alexa fluor 488/cy5 fluorescence ( k ) 
  are also shown .
yellow - to - orange staining indicates co - localized 
  socs-3 and dp-1 ( g and k ) .
socs-3 inhibits the transcriptional activity of 
  dp-1/e2f  because it was of interest to us to explore whether socs-3 
  actually would be able to regulate the transcriptional activity of dp-1 , we 
  next investigated the effect of socs-3 on this transcriptional activity in hek 
  293 cells .
as shown in a previous study 
  ( 35 ) , because expression of 
  the endogenous dp-1 is widely observed in several cell lines and the level of 
  dp-1 is considered to be sufficient to exhibit e2f / dp-1 transcriptional 
  activity , using the cyclin - e - luc assay we explored the effect of socs-3 on 
  endogenously expressed dp-1 in hek 293 cells .
first , we examined whether the 
  e2f / dp-1 transcriptional activity in the cells depended on endogenously 
  expressed dp-1 .
3a , lane 2 , cyclin - e - luc activity was 
  dramatically inhibited by dp-1 sirna .
these observations suggest that 
  endogenous dp-1 plays an important role in e2f / dp-1 transcriptional activity 
  in the cells .
therefore , we next addressed whether socs-3 could actually 
  inhibit e2f / dp-1-dependent transcriptional activity in an endogenous 
  dp-1-driven cyclin - e - luc activity .
as expected , scos-3 ( wt ) expression 
  inhibited about 50% of the transcriptional activity 
  ( fig .
however , such inhibitory effect was not observed with socs-3 
  ( 156172 ) , in which the dp-1-binding region is absent 
  ( fig .
importantly , because this inhibitory action of socs-3 was eliminated 
  completely by socs-3 sirna ( fig . 
 
3a , lane 6 ) , these results strongly suggest that 
  the inhibitory effect of socs-3 on endogenous dp-1-dependent transcriptional 
  activity is specific to socs-3 .
in addition , to support the contribution of endogenous dp-1 and exogenous 
  socs-3 , we investigated the expression of both proteins by western blotting . 
 
3b , 
  the endogenous dp-1 expression in hek 293 cells was almost completely 
  inhibited by dp-1 sirna . also , the expression of exogenous socs-3 at the 
  protein level in socs-3-expressing cells was inhibited by socs-3 sirna 
  ( fig .
next , using 
  the chip assay , we explored the interference of socs-3 with the binding of 
  dp-1/e2f-1 to the cyclin - e promoter .
together , these 
  results suggest to us the possibility that socs-3 may retard cell cycle 
  progression by inhibiting dp-1-elicited transcriptional activity of the 
  cyclin - e gene .
a , hek 293 cells were transiently transfected with the indicated 
  expression plasmids and sirna together with reporter ( cyclin - e - luc ) and 
  internal control ( prl - tk - luc ) plasmids .
after transfection , the cells were 
  incubated for 2430 h , and then cell extracts were prepared . 
 
firefly luciferase activity ( cyclin - e - luc ) from triplicate samples 
  was determined and normalized against renilla luciferase ( prl - tk - luc ) 
  activity .
effects of sirna were analyzed by western blotting with anti - dp-1 
  antibody ( b ) and anti - socs-3 antibody ( c ) .
d , chip 
  assays for binding of dp-1/e2f-1 to the cyclin - e promoter . hek 293 cells were 
  transiently transfected with the indicated expression plasmids and sirna and 
  then were fixed and subjected to the chip assay .
pcr amplification of dna 
  precipitated with anti - dp-1 ( lanes 37 ) and anti - e2f-1 
  ( lanes 812 ) using primers for the cyclin - e promoter . input 
  lane ( lane 1 ) shows products after pcr amplification of chromatin dna 
  before the addition of an antibody .
pcr products were separated by 2% agarose 
  gel electrophoresis and visualized by ethidium bromide staining .
a , hek 293 cells were transiently transfected with the indicated 
  expression plasmids and sirna together with reporter ( cyclin - e - luc ) and 
  internal control ( prl - tk - luc ) plasmids .
after transfection , the cells were 
  incubated for 2430 h , and then cell extracts were prepared . 
 
firefly luciferase activity ( cyclin - e - luc ) from triplicate samples 
  was determined and normalized against renilla luciferase ( prl - tk - luc ) 
  activity .
effects of sirna were analyzed by western blotting with anti - dp-1 
  antibody ( b ) and anti - socs-3 antibody ( c ) .
d , chip 
  assays for binding of dp-1/e2f-1 to the cyclin - e promoter . hek 293 cells were 
  transiently transfected with the indicated expression plasmids and sirna and 
  then were fixed and subjected to the chip assay .
pcr amplification of dna 
  precipitated with anti - dp-1 ( lanes 37 ) and anti - e2f-1 
  ( lanes 812 ) using primers for the cyclin - e promoter .
input 
  lane ( lane 1 ) shows products after pcr amplification of chromatin dna 
  before the addition of an antibody .
pcr products were separated by 2% agarose 
  gel electrophoresis and visualized by ethidium bromide staining .
a , hek 293 cells were transfected with the indicated expression 
  plasmids , cultured for 24 h , harvested , and stained with propidium iodide 
  using cycletest plus .
b , hek 293 cells were transiently transfected or not with the 
  indicated plasmids and sirna , and then cultured for 2 days in dmem containing 
  10% fetal bovine serum .
the number of viable cells was measured using the 
  celltiter - glo assay system based on quantification of the atp present .
a , hek 293 cells were transfected with the indicated expression 
  plasmids , cultured for 24 h , harvested , and stained with propidium iodide 
  using cycletest plus .
b , hek 293 cells were transiently transfected or not with the 
  indicated plasmids and sirna , and then cultured for 2 days in dmem containing 
  10% fetal bovine serum .
the number of viable cells was measured using the 
  celltiter - glo assay system based on quantification of the atp present .
socs-3 inhibits the cell cycle progression of dp-1-transfected 
  cells  to verify the above - mentioned possibility , we used flow 
  cytometry to determine whether or not socs-3 could retard the 
  e2f / dp-1-dependent cell cycle progression .
we transfected hek 293 cells with 
  the appropriate expression plasmids and then analyzed the distribution of 
  cells in the various phases of the cell cycle , as described previously 
  ( 27 ) .
4a , dp-1 enhanced 
  the proportion of hek 293 cells that had passed from g1 to s phase 
  ( fig .
furthermore , we examined cell growth by conducting a 
  luminescent cell viability assay , which is a homogeneous method for 
  determining the number of viable cells .
as we expected , the stimulated cell 
  growth of dp-1-transfected cells was clearly eliminated by dp-1 sirna 
  ( fig .
4b , lane 
  4 ) . when the cells were co - transfected with socs-3 and dp-1 expression 
  plasmids , although the dp-1-mediated cell growth was markedly inhibited by 
  socs-3 ( fig .
4b , 
  lane 5 ) , importantly , this inhibitory action of socs-3 was blocked by 
  socs-3 sirna ( fig . 
  4b , lane 6 ) .
these results suggest that socs-3 
  acts as a negative regulator of the cell cycle and cell growth under e2f / dp-1 
  control .
dp-1 eliminates the negative regulation of socs-3 in stat 
  signaling  finally , to confirm definitively this interaction between 
  socs-3 and dp-1 , in reverse , we explored whether dp-1 could eliminate the 
  negative regulation of socs-3 in jak - stat signaling . to explore this
, we 
  examined whether dp-1 could block the socs-3 inhibition of lif - stimulated 
  stat3 transcriptional activity by conducting a luciferase assay with a 
  reporter plasmid bearing the apre promoter and luciferase gene .
socs-3 ( wt ) 
  dramatically inhibited the lif - stimulated stat3 transcriptional activity in 
  hek 293 cells ( fig . 5 , lane 
  3 ) .
this inhibition was almost completely blocked by socs-3 sirna 
  ( fig . 5 , lane 4 ) . 
 
importantly , dp-1 clearly eliminated the inhibitory action of socs-3 toward 
  lif - stimulated stat3 transcriptional activity in socs-3 ( wt)-transfected cells 
  ( fig . 5 , lane 14 ) . in 
  addition , as we expected , this elimination by dp-1 was blocked by dp-1 sirna 
  ( fig . 5 , lane 16 ) . 
 
however , such elimination was not observed for socs-3 
  ( 156172)-mediated inhibition of the stat3 transcriptional 
  activity , because socs-3 ( 156172 ) is not able to bind to dp-1 
  ( fig . 5 , lane 18 ) . 
 
these results strongly indicate that dp-1 was able to block the socs-3 action 
  as a negative regulator of jak - stat signaling .
herein , using the two - hybrid system , we identified dp-1 as a 
  socs-3-interacting protein . because e2f / dp-1 plays an important role in the 
  g1-to - s phase transition in the cell cycle 
  ( 2224 )
, 
  we suspected that socs-3 may regulate cell cycle progression under e2f / dp-1 
  control via interaction with dp-1 .
although it is well known that 
  retinoblastoma tumor suppressor protein ( rb ) regulates negatively e2f function 
  by interacting directly with this transcriptional protein 
  ( 2931 , 
  3638 ) , 
  to our knowledge , a dp-1-interacting protein that is able to regulate cell 
  cycle progression under e2f / dp-1 control has not previously been demonstrated . 
 
our present study is the first one to show that socs-3 acts as a negative 
  regulator of the cell cycle under e2f / dp-1 control by interacting with dp-1 . 
 
interestingly , these findings also suggest the possibility that human socs-3 
  may regulate tumor cell growth and cell differentiation via this novel 
  mechanism .
figure 5.dp-1 eliminates the inhibitory action of socs-3 toward lif - stimulated 
  stat3 transcriptional activity .
hek 293 cells were transiently transfected 
  with various combinations of flag - socs-3 ( wt ) , flag - socs-3 
  ( 156172 ) , 6myc - dp-1 ( wt ) , and sirna in the presence of 
  the apre - luc and prl - tk - luc reporter plasmids .
the cells were incubated in the 
  presence or absence of 10 ng / ml hlif for 6 h , and then the cell extracts were 
  prepared .
firefly luciferase activity ( apre - luc ) from triplicate 
  samples was determined and normalized against renilla luciferase 
  ( prl - tk - luc ) activity .
dp-1 eliminates the inhibitory action of socs-3 toward lif - stimulated 
  stat3 transcriptional activity .
hek 293 cells were transiently transfected 
  with various combinations of flag - socs-3 ( wt ) , flag - socs-3 
  ( 156172 ) , 6myc - dp-1 ( wt ) , and sirna in the presence of 
  the apre - luc and prl - tk - luc reporter plasmids .
the cells were incubated in the 
  presence or absence of 10 ng / ml hlif for 6 h , and then the cell extracts were 
  prepared .
firefly luciferase activity ( apre - luc ) from triplicate 
  samples was determined and normalized against renilla luciferase 
  ( prl - tk - luc ) activity .
we found using an immunoprecipitation assay that socs-3 interacted with 
  dp-1 both in cells in vitro as well as in vivo 
  ( fig . 1 , b and 
  c ) .
in addition , using a series of deletion mutants of 
  socs-3 , we determined that the 156172 amino acid region of socs-3 was 
  required for interaction with dp-1 ( fig . 
 
although the function of this region located between 
  sh2 and socs - box domains is not yet known , this region is considered to be a 
  part of the domain interacting with dp-1 .
it is well known that dp-1 
  transcriptional activity is regulated by interaction with p53 
  ( 39 ) or arf 
  ( 40 , 
  41 ) and also that e2f is 
  controlled by interaction with prb 
  ( 2931 , 
  3638 ) . 
  however , because the cell cycle is a series of complicated cellular events 
  regulated by many kinds of signaling molecules , it was of much interest to us 
  to identify novel factors interacting with e2f / dp-1 and also to demonstrate 
  the regulatory mechanism involving e2f / dp-1 in detail .
therefore , we 
  investigated here the regulatory mechanism of socs-3 with respect to e2f / dp-1 
  control of the cell cycle .
several recent studies have shown that the 
  decreased expression of socs-1 , -2 , and -3 by hypermethylation of its gene 
  promoter is closely related to aggravation of some cancers 
  ( 1121 ) . 
  although these observations suggested to us some regulatory action of socs-1 
  for development and progression of tumor cells , the molecular mechanism 
  underlying the inhibitory action of socs family proteins
we found that socs-3 inhibited the transcriptional 
  activity of e2f / dp-1 via its interaction with dp-1 and consequently retarded 
  the cell cycle and cell growth under e2f / dp-1 control ( figs . 
  3 and 
  4 ) , though socs-3 did not 
  completely inhibit the transcriptional activity of e2f / dp-1 
  ( fig .
importantly , these inhibitions of socs-3 were almost completely blocked by 
  socs-3 sirna ( figs . 3 and 
  4b ) . as shown in 
  fig .
2 , the localization of 
  both dp-1 and socs-3 co - overexpressed in hek 293 cells almost was the same as 
  that of each overexpressed alone ; and , importantly , both proteins were 
  detected together in the cytoplasm . because we observed the co - localization of 
  dp-1 and socs-3 in the cytoplasm ( fig .
2 , 
  g and k ) , we suspect that socs-3 probably may 
  inhibit the transcriptional activity by interacting with dp-1 located in the 
  cytoplasm and promote the proteolysis of dp-1 via the socs - box .
this 
  possibility is supported by our data from the chip assay showing that socs-3 
  clearly inhibited dna binding activity of dp-1/e2f-1 at the cyclin - e promoter 
  ( fig .
figure 6.model illustrating the regulatory mechanisms between socs-3 and dp-1 for 
  jak - stat signaling and cell cycle progression .
several kinds of cytokines 
  induce socs-3 expression via the jak - stat signaling system , and then the 
  endogenous socs-3 inhibits the transcriptional activity of e2f / dp-1 by binding 
  to cytoplasmic dp-1 .
consequently socs-3 acts as a negative regulator of cell 
  cycle progression under e2f / dp-1 control . acting differently
, dp-1 eliminates 
  the inhibitory action of endogenous socs-3 toward jak - stat signaling .
model illustrating the regulatory mechanisms between socs-3 and dp-1 for 
  jak - stat signaling and cell cycle progression .
several kinds of cytokines 
  induce socs-3 expression via the jak - stat signaling system , and then the 
  endogenous socs-3 inhibits the transcriptional activity of e2f / dp-1 by binding 
  to cytoplasmic dp-1 .
consequently socs-3 acts as a negative regulator of cell 
  cycle progression under e2f / dp-1 control . acting differently
, dp-1 eliminates 
  the inhibitory action of endogenous socs-3 toward jak - stat signaling .
interestingly , because socs-1 and socs-2 also were able to interact with 
  dp-1 and to inhibit its transcriptional 
  activity,3 both socs 
  proteins also may act as novel negative regulator of e2f / dp-1 via direct 
  interaction with dp-1 .
( 26 ) identified a novel dp 
  subclass , dp-3 , that is able to inhibit e2f-1 transcriptional activity . 
 
therefore , our interest was to address whether socs-3 also is able to interact 
  with dp-3 .
consequently , we proved that socs-3 directly is able to interact 
  with dp-3 ( fig .
although it is very important to explore the regulatory action of 
  socs-3 for dp-3 , these results suggest to us a novel mechanism of socs 
  protein - mediated regulation of tumor cell growth and development . on the other hand , to confirm our finding that human socs-3 was an 
  interacting protein of dp-1 , approaching the question from another angle , we 
  investigated whether dp-1 could eliminate the inhibitory action of socs-3 
  toward lif - stimulated stat3 transcriptional activity in jak - stat signaling . 
 
consequently we observed that dp-1 almost completely eliminated such 
  inhibitory action of socs-3 in this signaling pathway 
  ( fig .
as described above , the 
  cytoplasmic dp-1 may interact with endogenous socs-3 induced by cytokines or 
  growth hormones and act as its modulator .
these observations lead us to 
  speculate that socs-3 and dp-1 may alternatively regulate each other in local 
  sites of tissues having inflammatory reaction such as tumor and rheumatoid 
  arthritis ( fig .
also , interestingly , it has been shown that socs-3 is deeply involved in 
  both initiation and development of allergic diseases such as atopic dermatitis 
  and asthma ( 42 ) .
therefore , 
  dp-1 may act as a potent modulator of allergic diseases(s ) regulated by 
  socs-3 .
importantly , it has been shown that ubiquitinated dp-1 is degraded by 
  the proteasome system ( 43 ) . 
 
also , socs-3 is known to recruit ecs ( elongin b / c - cul2/cul5-socs - box protein ) 
  e3 ligase , resulting in accelerated proteasomal degradation of target proteins 
  and socs-3 
  ( 4447 ) . 
  therefore , to understand how each of these factors actually exhibits its 
  regulatory action in the cytoplasm , in further experiments , it will be very 
  important for us to address in detail the degradation events of both factors 
  in the cytoplasm . in conclusion
, we demonstrated that human socs-3 interacted with dp-1 and 
  regulated cell cycle progression under e2f / dp-1 control 
  ( fig .
thus , these findings 
  provide us with new insights into the role of socs-3 in cell growth and cell 
  differentiation besides its function as a negative regulator of jak - stat 
  signaling .
 recent studies using socs family knock - out mice have suggested that socs 
  proteins have multiple biological functions in addition to their role as 
  negative regulators of jak - stat signaling . to explore these other functions of 
  this family of proteins , we used yeast two - hybrid screening to find proteins 
  interacting with human socs-3 . 
 we identified the transcriptional factor dp-1 
  as a socs-3-interacting protein involved in regulation of the cell cycle . 
  
 immunoprecipitation - western blot assay showed that this interaction between 
  these endogenous proteins occurred in cells both in vitro and in 
  vivo . 
 socs-3 interacted with the c - terminal region of dp-1 , and amino 
  acids 156172 of socs-3 were required for this interaction . 
 confocal 
  microscopy revealed that socs-3 and dp-1 were primarily colocalized in the 
  cytoplasm . 
 socs-3 inhibited e2f / dp-1 transcriptional activity under the 
  cyclin - e promoter and actually inhibited cell cycle progression and cell 
  growth under e2f / dp-1 control . in contrast , dp-1 almost completely eliminated 
  the inhibitory action of socs-3 on lif - stimulated stat-3 transcriptional 
  activity in jak - stat signaling . 
 interestingly , the alternative regulatory 
  action of socs-3 and dp-1 was dramatically eliminated by each sirna . taken 
  together , 
 these findings demonstrate that socs-3 acts as a negative regulator 
  of the cell cycle progression under e2f / dp-1 control by interfering with 
  heterodimer formation between dp-1 and e2f and also that dp-1 plays an 
  important role in controlling jak - stat signaling . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION