cell lines , cultures and transfections : a madin - darby bovine kidney cell 
 line ( mdbk ) ( hsrrb , osaka , japan ) was cultured in dulbecco s modified eagle s medium ( dmem ) 
 with 10% fetal calf serum ( fcs ) . a human cervical carcinoma cell line ( hela )
( riken cell 
 bank , tsukuba , japan ) was cultured as described in previous studies [ 10 , 14 ] .
a cattle xlf gene 
 ( nm_001075393.1 ) with an artificial ecori site at the 5 end and bamhi site at the 3 end 
 was synthesized .
the fragment was confirmed by sequencing and ligated to the ecori and bamhi 
 sites of the peyfp - c1 vector to give the in - frame fusion gene .
peyfp - cattle xlf , 
 peyfp - cattle xlf ( 162299 ) , peyfp - cattle xlf ( 162287 ) or peyfp - c1 was transient transfected 
 in cells using fugene hd ( promega , madison , wi , u.s.a . )
the cells were cultured for 2 days and then monitored under an fv300 confocal 
 laser scanning microscope ( olympus , tokyo , japan ) as previously described [ 9 , 11 , 12 ] .
immunoblotting : the extraction of total lysates and western blot analysis 
 were conducted based on the previous methods [ 11 , 
 13 ] .
the membranes 
 were blocked in blocking one ( nacalai tesque , kyoto , japan ) for 30 min .
the following 
 antibodies were used : a rabbit anti - xlf polyclonal antibody ( a300 - 730a ) ( bethyl 
 laboratories , montgomery , tx , u.s.a . ) , a rabbit anti - gfp polyclonal antibody ( fl ) ( santa 
 cruz biotechnology , santa cruz , ca , u.s.a . ) , a mouse anti - ku70 monoclonal antibody ( n3h10 ) 
 ( neomarkers , fremont , ca , u.s.a . ) or a mouse anti--actin monoclonal antibody ( sigma , st . 

three antibodies ( i.e. , anti - xlf antibody , anti - ku70 antibody and 
 anti - gfp antibody ) were diluted in signal enhancer hikari ( nacalai tesque ) , respectively . 

the binding to each protein was visualized using a select western blotting detection system 
 ( ge healthcare bio - sci .
piscataway , nj , u.s.a . ) , in accordance with the manufacturer s 
 instructions .
immunofluorescence staining : immunofluorescence staining was conducted as 
 previously described [ 9 , 11 ] .
briefly , the fixed cells were blocked for 10 min using a blocking 
 solution and then incubated for 30 min at room temperature with a mouse anti-h2ax 
 monoclonal antibody ( jbw301 ) ( upstate biotechnology inc . , charlottesville , va , u.s.a . ) or a 
 rabbit anti - xlf polyclonal antibody ( x4754 ) ( sigma ) . after washing with pbs , detection of 
 each protein
was performed using alexa fluor 568-conjugated secondary antibodies ( molecular 
 probes , eugene , or , u.s.a . ) .
local dna damage induction using laser and cell imaging : local dna damage 
 induction using laser and cell imaging was conducted as described previously [ 9 , 11,12,13 ] .
briefly , a 
 530% power scan ( for 1 sec ) from a 405 nm laser was used to induce local dsbs .
images of 
 living cells or fixed cells expressing eyfp - tagged cattle proteins or eyfp alone were 
 obtained using an fv300 confocal scanning laser microscopy system ( olympus ) .
expression and localization of cattle xlf in cattle cells : we examined the 
 expression and subcellular localization of xlf in cattle cells .
first , we examined the 
 expression of xlf and ku70 in the cattle cell line mdbk and the human cell line hela by 
 western blot analysis using the anti - xlf antibody and anti - ku70 antibody .
( a ) total cell lysates from each 
 cell line ( mdbk , 50 g ; hela , 10 g ) were analyzed 
 by western blotting using an anti - xlf antibody , an anti - ku70 antibody or an 
 anti--actin antibody .
( b , c ) subcellular localization of xlf in cattle ( mdbk ) cells 
 during the cell cycle .
the images shown 
 are a representative example for interphase cells or mitotic phase cells ( c ) .
these results demonstrate that the core nhej factors , xlf 
 and ku70 , are expressed in cattle cells .
total cell lysates from each 
 cell line ( mdbk , 50 g ; hela , 10 g ) were analyzed 
 by western blotting using an anti - xlf antibody , an anti - ku70 antibody or an 
 anti--actin antibody .
( b , c ) subcellular localization of xlf in cattle ( mdbk ) cells 
 during the cell cycle .
the images shown 
 are a representative example for interphase cells or mitotic phase cells ( c ) . to elucidate the localization of xlf in cattle cells
, we studied the distribution of xlf by 
 confocal laser microscopy ( fig . 1b and 1c ) . 

indirect immunofluorescence staining using the anti - xlf antibody showed that fluorescence 
 was detected in the nucleoplasm of mdbk cells during the interphase . on the other hand ,
the 
 fluorescence was detected throughout the cytoplasm of mdbk cells during the mitotic phase , 
 but not in the condensed chromosomes of the mitotic cells .
these observations indicate that 
 the localization of cattle xlf changes dynamically during the cell cycle .
to clarify the 
 localization of xlf in living cattle cells during the interphase , we examined the expression 
 and localization of eyfp - cattle xlf in mdbk cells .
the expression vector peyfp - c1 containing cattle xlf 
 ( peyfp - cattle xlf ) was transfected into mdbk cells ( fig .
( a ) schematics of eyfp - cattle 
 xlf chimeric protein and control protein ( eyfp ) .
( b ) extracts from cattle ( mdbk ) cells 
 transiently expressing the eyfp - cattle xlf or eyfp prepared and subjected to western 
 blotting using the anti - xlf , anti - gfp or anti--actin antibody .
living mdbk cells transiently expressing 
 eyfp - cattle xlf or eyfp were analyzed by confocal laser microscopy .
eyfp images for 
 the same cells are shown alone ( left panel ) or merged ( right panel ) with differential 
 interference contrast images ( dic ) ( center panel ) . ) .
2b , a signal of 
 eyfp - cattle xlf was detected in the transfectants by western blot analysis using the 
 anti - xlf antibody and anti - gfp antibody . by confocal laser microscopy
, we clarified that 
 eyfp - cattle xlf was localized in the nuclei of living interphase cells in eyfp - cattle xlf 
 transfectants ( fig . 2c ) .
expectedly , in eyfp 
 transfectants , we confirmed that eyfp was distributed throughout the cell excluding the 
 nucleolus ( fig . 2c ) .
( a ) schematics of eyfp - cattle 
 xlf chimeric protein and control protein ( eyfp ) .
( b ) extracts from cattle ( mdbk ) cells 
 transiently expressing the eyfp - cattle xlf or eyfp prepared and subjected to western 
 blotting using the anti - xlf , anti - gfp or anti--actin antibody .
living mdbk cells transiently expressing 
 eyfp - cattle xlf or eyfp were analyzed by confocal laser microscopy .
eyfp images for 
 the same cells are shown alone ( left panel ) or merged ( right panel ) with differential 
 interference contrast images ( dic ) ( center panel ) .
eyfp - cattle xlf accumulates quickly at dsbs induced by laser 
 microirradiation : we examined whether eyfp - cattle xlf accumulates quickly at the 
 405 nm laser - induced dsb sites ( fig . 3afig .
( a ) 
 the localization and accumulation of eyfp - cattle xlf at dsbs induced by 405 nm laser 
 irradiation were examined .
( b ) imaging of living eyfp - cattle xlf - transfected mdbk 
 cells before ( upper panel ) and at 1 min after ( lower panel ) microirradiation . left 
 panel , eyfp - cattle xlf ; right panel , differential interference contrast images ( dic ) . 

( c ) immunostaining of microirradiated 
 eyfp - cattle xlf - transfected cells with anti-h2ax antibody .
the cells were fixed and 
 stained with the anti- h2ax antibody at 5 min postirradiation . left panel , 
 eyfp - cattle xlf ; center panel , h2ax image ; right panel , merged image .
( d ) 
 time - dependent eyfp - cattle xlf accumulation in living cells ( 5 - 120 sec ) after 
 irradiation .
upper panel , eyfp - cattle xlf ; lower panel , differential interference 
 contrast images ( dic ) . ) . as shown in fig .
3b , we observed that 
 eyfp - cattle xlf accumulated at the microirradiated sites in the living cattle cells .
next , 
 we investigated whether cattle xlf accumulated at 405 nm laser - induced dsb sites by 
 immunostaining with an antibody that detects h2ax . as shown in fig .
3c , eyfp - cattle xlf colocalized with the dsb marker h2ax at 
 microirradiated sites in mdbk cells .
next , we carried out time - lapse imaging of eyfp - cattle 
 xlf - transfected mdbk cells .
3d , 
 we observed eyfp - cattle xlf accumulation at the microirradiated sites 5 sec after 
 irradiation . in eyfp - cattle xlf - transfected cells ,
these results reveal that after irradiation , 
 eyfp - cattle xlf quickly accumulates and formes foci at laser - induced dsbs in living 
 cells .
( a ) 
 the localization and accumulation of eyfp - cattle xlf at dsbs induced by 405 nm laser 
 irradiation were examined .
( b ) imaging of living eyfp - cattle xlf - transfected mdbk 
 cells before ( upper panel ) and at 1 min after ( lower panel ) microirradiation . left 
 panel , eyfp - cattle xlf ; right panel , differential interference contrast images ( dic ) . 

( c ) immunostaining of microirradiated 
 eyfp - cattle xlf - transfected cells with anti-h2ax antibody .
the cells were fixed and 
 stained with the anti- h2ax antibody at 5 min postirradiation . left panel , 
 eyfp - cattle xlf ; center panel , h2ax image ; right panel , merged image .
( d ) 
 time - dependent eyfp - cattle xlf accumulation in living cells ( 5 - 120 sec ) after 
 irradiation .
upper panel , eyfp - cattle xlf ; lower panel , differential interference 
 contrast images ( dic ) .
the c - terminal region ( ctr ) of cattle xlf is essential for the nuclear localization 
 and recruitment of xlf to dsbs in cattle cells : to determine the region essential 
 for nuclear localization of cattle xlf , we investigated the localization of cattle xlf and 
 its mutant .
firstly , the peyfp - cattle xlf and its mutants were transfected into mdbk cells . 
 as shown in fig .
4.the c - terminal region ( ctr ) is vital for the nuclear localization and recruitment of 
 cattle xlf to dsbs in vivo .
( a ) extracts from cattle ( mdbk ) cells 
 transiently expressing the indicated cattle xlf deletions were prepared and subjected 
 to western blotting using the anti - gfp or anti--actin antibody .
( b , c ) identification 
 of essential domain of cattle xlf for nuclear localization and for accumulation at 
 dsbs .
the localization 
 and accumulation of the chimeric proteins at laser - induced dsbs were investigated via 
 live cell imaging .
the results are summarized on the right : cellular localization ( n , 
 nucleus ; c , cytoplasm ) and formation of focus ( + , accumulated at microirradiated 
 sites ; - , not accumulated at microirradiated sites ) .
( d ) immunostaining of microirradiated eyfp - cattle xlf 
 ( 162287)-transfected cells with anti-h2ax antibody .
the cells were fixed and stained 
 with the anti-h2ax antibody at 5 min postirradiation . left panel , eyfp - cattle xlf 
 ( 162 - 287 ) ; center panel , h2ax image ; right panel , merged image .
( e ) identity between 
 the cattle xlf and human xlf at the amino acid level and the ctr of cattle xlf ( amino 
 acids 288299 ) .
( f ) the alignment of the primary sequence among homologous xlf 
 proteins . for comparison , the basic ( red ) or non - basic residues ( black )
, a signal of each eyfp - cattle xlf mutant was detected in the extracts of each 
 transfectant by western blot analysis using the anti - gfp antibody . by confocal laser 
 microscopy
, we confirmed that eyfp - cattle xlf was localized in the nuclei of living 
 interphase cells .
we observed that n - terminal deletion mutant eyfp - cattle xlf ( 162299 ) 
 localized predominantly in the nuclei , whereas eyfp - cattle xlf ( 162287 ) as well as eyfp was 
 distributed throughout the cell excluding the nucleolus in mdbk cells ( figs . 2c , 4b and 4c ) .
these results indicate that 12 c - terminal amino 
 acids ( amino acids 288299 ) of cattle xlf are vital for the nuclear localization of xlf in 
 cattle cells ( fig .
which region 
 of cattle xlf is essential for its accumulation at dsbs in vivo , we tested 
 whether the xlf mutant proteins could be recruited to dsbs induced by microirradiation .
we 
 observed that a n - terminal deletion mutant eyfp - cattle xlf ( 162299 ) as well as the wild 
 type eyfp - cattle xlf , accumulated at the dsbs sites in the living mdbk cells ( fig . 3c and 
 data not shown ) . on the other hand , the mutant protein eyfp - cattle xlf ( 162287 ) failed to 
 accumulate at the dsbs sites , which was detected by the dsb marker h2ax ( fig . 4c and 4d ) , indicating that deletion of the 
 c - terminal end 12 amino acids abolished the recruitment of cattle xlf to dsbs .
the cattle 
 and human xlf proteins are 81.9% identical at the amino acid level , whereas the c - terminal 
 domain ( amino acids 220299 ) of cattle xlf retains only 56.4% identity to human ( fig .
interestingly , we confirmed that the basic 
 amino acids in the ctr of xlf are evolutionarily highly conserved among humans and domestic 
 animal species , e.g. , cattle , goats , horses and avian , but not in yeast ( fig .
4f and data not shown ) , which strongly suggests 
 the biological significance of the xlf ctr in domestic animals .
the c - terminal region ( ctr ) is vital for the nuclear localization and recruitment of 
 cattle xlf to dsbs in vivo . ( a )
extracts from cattle ( mdbk ) cells 
 transiently expressing the indicated cattle xlf deletions were prepared and subjected 
 to western blotting using the anti - gfp or anti--actin antibody .
( b , c ) identification 
 of essential domain of cattle xlf for nuclear localization and for accumulation at 
 dsbs .
the localization 
 and accumulation of the chimeric proteins at laser - induced dsbs were investigated via 
 live cell imaging .
the results are summarized on the right : cellular localization ( n , 
 nucleus ; c , cytoplasm ) and formation of focus ( + , accumulated at microirradiated 
 sites ; - , not accumulated at microirradiated sites ) .
( d ) immunostaining of microirradiated eyfp - cattle xlf 
 ( 162287)-transfected cells with anti-h2ax antibody .
the cells were fixed and stained 
 with the anti-h2ax antibody at 5 min postirradiation . left panel , eyfp - cattle xlf 
 ( 162 - 287 ) ; center panel , h2ax image ; right panel , merged image .
( e ) identity between 
 the cattle xlf and human xlf at the amino acid level and the ctr of cattle xlf ( amino 
 acids 288299 ) .
( f ) the alignment of the primary sequence among homologous xlf 
 proteins . for comparison , the basic ( red ) or non - basic residues ( black )
to develop next - generation chemotherapeutics for cancer and other disease is important to 
 clarify the molecular mechanisms of c - nhej .
human xlf is the most recently identified core 
 nhej factor , and it appears to play essential roles in c - nhej .
expectedly , xlf - deficient cells derived from human patients and from 
 knockout mice show ionizing radiation sensitivity [ 3 , 
 15 ] . in addition
, sirna - mediated downregulation of 
 xlf in human cell lines leads to radiosensitivity and impaired nhej . .
homologues of the xlf gene were predicted in several eukaryotic 
 organisms . on the other hand , the expression , 
 function and regulation mechanism of xlf
have not been elucidated in animal species other 
 than mice and humans [ 1 , 3 , 11 , 16 , 21 ] .
domestic cattle are 
 important domestic animals as livestock and draft animals in not only japan , but also many 
 countries .
recently , cattle have been an ideal animal model for assessing chronic radiation 
 exposure [ 5 , 20 ] . however , the molecular mechanism of c - nhej is still unknown in cattle cells . in 
 this study
, we examined the expression and subcellular localization of cattle xlf and its 
 mutants in cattle cell line mdbk .
we found that xlf as well as other core nhej protein ku70 
 is expressed in cattle cells , and the localization of cattle xlf changes dynamically during 
 the cell cycle .
in addition , xlf might play a vital role in the repair of dsb immediately 
 after microirradiation of cattle cells .
moreover , our data showed that the ctr of cattle xlf 
 is vital for the nuclear localization of xlf and for the accumulation of xlf at dsbs 
 in vivo .
these findings suggest that the mechanisms regulating of the 
 localization and recruitment to dsbs play a key role in the function of cattle xlf .
cattle xlf ( nm_001075393.1 ) as well as goat xlf ( xp_005676612.1 ) and sheep xlf 
 ( xp_004004987.1 ) is a 299-amino acid protein .
the cattle and goat xlf genes are 94.6% 
 identical at the amino acid level .
in addition , the cattle and sheep xlf genes are 94.3% 
 identical at the amino acid level . on the other hand ,
human xlf is a 299-amino acid protein , which contains an n - terminal head 
 domain ( amino acids 1141 ) , a coiled - coil central domain ( amino acids 142230 ) and a non 
 structured c - terminal domain ( amino acids 231299 ) [ 1 , 
 2 , 17 ] . 

comparison with other eukaryotic homologues shows a high degree of sequence similarity 
 within the 220 n - terminal amino acids , while the c - terminal domain ( amino acids 225299 ) is 
 less conserved [ 1 , 2 , 17 ] . on the basis of experimental 
 findings
, there are some reports concerning the role of the c - terminal domain of xlf in 
 humans , but not in animals including cattle .
yano et al . reported that a 
 10-amino - acid deletion at the c - terminal end completely abolishes the ku - xlf interaction and 
 the accumulation of xlf at dsbs . on the other 
 hand , malivert et al .
have reported that the c - terminal end ( amino acids 
 231299 ) of human xlf is dispensable for dna repair in vivo . in this study
, our data showed that a 12-amino - acid 
 deletion at the c - terminal end abolishes the accumulation of cattle xlf at dsbs . altogether
, 
 we conclude that the xlf ctr is important for the accumulation of xlf at dsbs in both human 
 and cattle cells , although the role of the c - terminal region of xlf remains controversial in 
 human cells .
it was demonstrated that there is a general absence of conservation in the 75 c - terminal 
 amino acids among humans and other species , although the extreme c terminus of xlf contains 
 a small conserved basic cluster , which was proposed as a putative nls ( krkk ) [ 1 , 2 ] .
our data 
 revealed experimentally that the ctr of cattle xlf is critical for the nuclear localization 
 of xlf and recruitment to dsbs , whereas the n - terminal domain is not essential .
in addition , 
 basic amino acids in the ctr of cattle xlf are evolutionarily conserved among ctr of 
 domestic animals , which suggests the common biological significance of the xlf ctr in 
 domestic animals .
we consider that there is only one nls ( kvkrkklr ) in cattle xlf , and the 
 nls is a classical monopartite nls having a single cluster of basic amino acid residues .
we 
 speculate that the c - terminus of 75 amino acids is important for a specific function in each 
 species , whereas the xlf ctr is critical for the regulation of common functions in domestic 
 animals .
, we showed that xlf is expressed in cattle cells and the localization of 
 cattle xlf changes dynamically during the cell cycle .
in addition , our data showed that the 
 localization and recruitment of cattle xlf to dsb sites at an early stage following 
 irradiation are dependent on the ctr .
these basic informations might be useful to develop 
 the molecular - targeting therapeutic drug taking xlf as a target molecule for human and 
 domestic animals .
further studies to elucidate the mechanisms regulating cattle xlf at dsbs 
 will lead to a better understanding of the physiological function of xlf not only in cattle 
 cells , but also in cells of human and other domestic animals .
inherited mutations of core 
 c - nhej factors ( e.g. , dna - pkcs , dna ligase iv and xlf ) , have been discovered in humans 
 . on the other hand , inherited
mutations of the 
 dna - pkcs , which cause scid , have been identified in not only humans , but also domestic 
 animals , i.e. , mice , horses or dogs .
therefore , 
 further comparative studies might provide available information for the development of new 
 clinical medicines and new chemoradiotherapies for humans and domestic animals including 
 cattle .
 clinically , many chemotherapeutics and ionizing radiation ( ir ) have been 
 applied for the treatment of various types of human and animal malignancies . 
 these 
 treatments kill tumor cells by causing dna double - strand breaks ( dsbs ) . 
 core factors of 
 classical nonhomologous dna - end joining ( c - nhej ) play a vital role in dsb repair . 
 thus , it 
 is indispensable to clarify the mechanisms of c - nhej in order to develop next - generation 
 chemotherapeutics for cancer . the xrcc4-like factor ( xlf ; also called cernunnos or nhej1 ) 
 
 is the lastly identified core nhej factor . 
 the localization of core nhej factors might 
 play a critical role in regulating nhej activity . 
 the localization and function of xlf 
 have not been elucidated in animal species other than mice and humans . 
 domestic cattle 
 ( bos taurus ) are the most common and vital domestic animals in many 
 countries . here 
 , we show that the localization of cattle xlf changes dynamically during 
 the cell cycle . 
 furthermore , eyfp - cattle xlf accumulates quickly at microirradiated sites 
 and colocalizes with the dsb marker h2ax . 
 moreover , nuclear localization and accumulation 
 of cattle xlf at dsb sites are dependent on 12 amino acids ( 288299 ) of the c - terminal 
 region of xlf ( xlf ctr ) . 
 furthermore , basic amino acids on the xlf ctr are highly 
 conserved among domestic animals including cattle , goat and horses , suggesting that the 
 ctr is essential for the function of xlf in domestic animals . 
 these findings might be 
 useful to develop the molecular - targeting therapeutic drug taking xlf as a target molecule 
 for human and domestic animals . 
MATERIALS AND METHODS
RESULTS
DISCUSSION