animal : icr mice obtained from kyudo co. ( saga , japan ) were used in this 
 study . mice were housed in standard polypropylene cages in a temperature - controlled room 
 ( 22c ) with a 12 hr light , 12 hr dark cycle . studies were performed according to protocols for animal use , approved 
 by the yamaguchi university animal experimental guidelines ( permission number : 239 ) . sampling : icr female mice of 8 weeks old were mated with male mice of the 
 same strain . the day when a vaginal plug was detected was considered day 1 of gestation . mice were sacrificed on gestational days 12 , 14 and 16 ( d12 , d14 and d16 ) under avertin 
 anesthesia . the right uterine horn was removed , and each placenta was separated to labyrinth 
 zone ( lz ) and decidual basalis ( db ) . for the studies of effects of starvation , immunohistochemistry : sections of 4 m in thickness were prepared . after that , immunohistochemistry of map1lc3a 
 ( q9h492 , epitomics , san francisco , ca , u.s.a . ) , map1lc3b ( 18725 - 1-ap , proteintech , chicago , 
 il , u.s.a . ) and map1lc3c ( ap1804a , abgent , san diego , ca , u.s.a . ) was performed . deparaffinized sections for lc3a were autoclaved in citrate buffer solution ph 6.0 for 
 activation . next , all deparaffinized sections were treated with 0.3% 
 h2o2/methanol for 30 min to inactivate intrinsic peroxidase . sections were preblocked with 10% goat serum ( 426042 , nichirei , tokyo , japan ) for 30 min 
 before being incubated with specific antibodies described above at 4c overnight . after 
 that , sections were treated with simplestain max - po ( r ) ( 424141 , nichirei ) for 30 min . the 
 immunoreactions were visualized with dab ( d006 , dojindo , kumamoto , japan ) . each diluted concentration is as follows : lc3a ( 1:100 ) , lc3b 
 ( 1:100 ) and lc3c ( 1:100 ) . western blotting : map1lc3a , map1lc3b , map1lc3c , p53 ( sc-6243 , santa cruz 
 biotechnology , santa cruz , tx , u.s.a . ) and p70s6k ( bs-3498r , bioss , woburn , ma , u.s.a . ) each protein was extracted with 
 laemmli sample buffer ( 161 - 0737 , bio - rad , hercules , ca , u.s.a . ) according to the 
 manufacturer s protocol . total protein samples were resolved by 8 and 15% polyacrylamide gels 
 and transferred to immobilon - p membrane ( iseq00010 , millipore corporation , bedford , ma , 
 u.s.a . ) . membranes were blocked in pvdf blocking reagent for can get signal ( nypbr01 , 
 toyobo , osaka , japan ) for 1 hr at room temperature and then incubated with primary 
 antibodies at 4c overnight . membranes were washed and incubated with appropriate secondary 
 antibodies ( santa cruz biotechnology ) . actin ( sc-1616 , santa cruz biotechnology ) volumes 
 were also measured as a loading control . primary antibody and secondary antibody were 
 diluted with can get signal immunoreaction enhancer solution 1 and 2 , respectively ( nkb-201 
 and nkb301 , toyobo ) . each diluted concentration is as follows : lc3a ( 1:1,500 ) , lc3b 
 ( 1:2,000 ) , lc3c ( 1:1,000 ) , p53 ( 1:1,500 ) , p70s6k ( 1:1,500 ) , actin ( 1:4,000 ) and secondary 
 antibody ( 1:20,000 ) . bands were visualized by immunostar ld ( 296 - 69901 , wako , osaka , japan ) 
 in las3000 ( fujifilm , tokyo , japan ) . real - time rt pcr : total rna was prepared from each sample with isogen ii ( 311 - 07361 , nippon 
 gene , tokyo , japan ) . real - time pcr was performed in triplicate with power sybr green pcr master mix 
 ( 4267659 , applied biosystems , carlsbad , ca , u.s.a . ) in the step one real - time pcr systems 
 ( applied biosystems ) according to the manufacturer s instructions . used primers were as follows : dram ( forward : ttggattggtgggatgcat , reverse : 
 gcacggctaactcctggaagt ) , p53 ( forward : tgcatggacgatctgttgct , reverse : ttcacttgggccttcaaaaaa ) , 
 mtor ( forward : acagcccgtcacaatgca , reverse : gctacccgaatcagctcttca ) , mdm2 ( forward : 
 ggaggaaacgcaggacaaag , reverse : cgatggcattcagggagaag ) , lkb1 ( forward : acggcctggaatacctacaca , 
 reverse : gttgcccggcttgatgtc ) , ampk ( forward : tcgtgccgccccttt , reverse : ggtcagcatgcccacaaaa ) , 
 akt1 ( forward : tcacgccgcctgatcaa , reverse : cggcctccgctcactgt ) , pi3k ( forward : 
 cgagggagcaggaacatca , reverse : tgcggcatagacagtgaactg ) and gapdh ( forward : 
 catggccttccgtgttccta , reverse : gcggcacgtcagatcca ) statistics : numbers of used samples were three for each . statistical 
 analyses were performed using kruskall - wallis test and mann - whitney u test as appropriate . first , we investigated expression volumes of p53 and lc3 family during normal placentation 
 by western blotting ( fig . 
 1fig . 1.western blotting analysis for p53 , lc3a , b and c conducted on placental proteins . each lane represents labyrinth zone ( lz ) and decidua basalis ( db ) at d12 , d14 and d16 , 
 respectively . lca and b in db were significantly higher in expression volume than those in lz , 
 while lc3c showed no differences . lc3a and b tended to increase at d14 , whereas lc3c tended 
 to decrease at d14 ( lc3a lz p=0.027 , lc3b lz p=0.066 , lc3c 
 lz p=0.039 . western blotting analysis for p53 , lc3a , b and c conducted on placental proteins . each lane represents labyrinth zone ( lz ) and decidua basalis ( db ) at d12 , d14 and d16 , 
 respectively . next , we investigated changes of lc3 family induced by starvation ( fig . 2fig . 2.western blotting for p70s6k , lc3a , b and c in the normal placenta and placenta under 
 starvation . each lane represents labyrinth zone ( lz ) and decidua basalis ( db ) at d12 , 
 d14 and d16 , respectively . this suggests starvation induced mtor suppression and caused 
 autophagy . in association with p70s6k changes , 2 ) , while lc3a showed 
 significant increase only in db at d14 ( fig . lc3c was significantly increased at d12 and d14 , but significantly decreased at d16 ( fig . western blotting for p70s6k , lc3a , b and c in the normal placenta and placenta under 
 starvation . each lane represents labyrinth zone ( lz ) and decidua basalis ( db ) at d12 , 
 d14 and d16 , respectively . 3.summary of immunohistochemistry of lc3a , b and c. each positive cell and negative 
 cell is indicated by + and  , respectively . + / indicates that both positive and negative cells are 
 observed .. while syncytiotrophoblast cells expressed lc3a , b and c , giant 
 trophoblast cells expressed lc3c , but not lc3a . lc3c expressions in spongiotrophoblast cells 
 were positive at d12 and d16 , but negative at d14 . lc3b was observed in several giant 
 trophoblast cells at d14 , but lc3 negative giant trophoblast cells were also observed . then , we examined the changes of expression 
 region under the starvation . in syncytiotrophoblast cells , lc3a expression was vanished by 
 starvation , but not lc3b and c ( fig . a - c shows d14 , and d - f shows d14 under starving . ) . summary of immunohistochemistry of lc3a , b and c. each positive cell and negative 
 cell is indicated by + and  , respectively . a - c shows d14 , and d - f shows d14 under starving . finally , to reveal what signal caused p70s6k decrease and lc3 changes under starvation , we 
 chased changes in the mrna expression of genes related to glucose starvation and growth 
 factor . 5.real time rt - pcr for p53 , dram , mdm2 , lkb1 , ampk , pi3k and akt1 conducted in normal 
 placenta and placenta under starvation . p=0.05 .. at d12 , except for lkb1 in lz and akt1 in db , p53 , dram , mdm2 , lkb1 , ampk , pi3k and 
 akt1 showed significant increase in starving condition ( fig . although there was no significance , lkb1 in lz and akt1 
 in db also tended to be increased . on the other hand , real time rt - pcr for p53 , dram , mdm2 , lkb1 , ampk , pi3k and akt1 conducted in normal 
 placenta and placenta under starvation . this study revealed that ( i ) lc3a and b in db were expressed higher than those in lz , ( ii ) 
 lc3a , b and c expression patterns were different among each cell , ( iii ) starving induced 
 increase in lc3b expression specifically , and ( iv ) glucose starvation and growth factor 
 related genes were changed by starvation during normal placentation . these results suggest 
 autophagy mediated by each lc3 family is functionally distinct . in normal placenta , lc3a , b and c in lz at d14 showed differences from d12 and d16 . it was 
 reported that hif-1 decreases trophoblast invasion via autophagy , suggesting that autophagy is related to trophoblast invasion at d14 . lc3a expression in breast cancer cells is 
 stronger than normal breast cell   and important 
 for tumor suppression . autophagy works in both tumor suppression 
 and tumor growth [ 16 , 36 ] . under the starving condition , although autophagy as a life 
 support system may work for tumor growth , lc3 that reacts strongly against starvation in 
 this study was lc3b , but not lc3a . this may be occurred 
 for unlocking the autophagic brake of trophoblast invasion to recover the placental growth 
 retardation induced by starvation . additionally , excessive invasion of trophoblast could be 
 prevented by high lc3a expression in decidual cells during normal placentation . starving condition inactivated mtor , as reflected by reduced p70s6k expression . in 
 association with this , lc3b showed significant increase . women 
 with intrauterine growth restriction have higher lc3b protein levels than women with normal 
 pregnancy . still , lc3b expressions in these reports were related 
 to dram increase , but not via mtorc1 suppression . in our study , while dram showed 
 significant increase at d12 , it had no changes at d14 and d16 . although there is no doubt 
 that lc3b is related to autophagy via dram , our results suggest autophagy via mtorc1 is also 
 mediated by lc3b . lc3c showed significant increase at d12 and d14 , whereas it showed significant decrease at 
 d16 under starving condition . the activation of autophagy during infection provides 
 cell - autonomous protection through lysosomal degradation of invading pathogens , and there are reports that lc3c is necessary for 
 antibacterial autophagy [ 20 , 34 ] . although there are significances , lc3c changes were slight when 
 compared with lc3a and b. lc3c would mainly mediate antibacterial autophagy and would not 
 have an important role for placental growth and starvation . nonetheless , there is a 
 possibility that lc3c works for compensatory mechanism against other lc3 family . as signal pathways controlling mtorc1 during starvation , insulin , starvation induced pi3k and akt1 increase at d12 . although it suggests this signal is 
 related to mtorc1 expression at d12 under starvation , this signal activates mtorc1 and 
 suppresses autophagy at ordinary times . nonetheless , considering to p70s6k decreasing , 
 general gene expression seems to move for mtorc1 suppression . when glucose is exhausted , atp production is suppressed , and ampk is activated via lkb1 . p53 and dram were also increased with lkb1 and ampk at 
 d12 , so glucose starvation  lkb1  ampk  p53  dram  autophagy pathway might work at 
 d12 . for the reason of the differences observed at lkb1 and ampk at d12 and d16 , premature 
 placental angiogenesis and insufficient crosstalk at d12 are conceivable . lkb1 and ampk decrease observed at d16 could be for the 
 prevention of the excessive autophagy induced by this signal . we suspect these 
 differences were the result from each cell metabolism , function and cell cycle . nutrients 
 starvation could not affect localization of lc3 family expression , except for lc3a in 
 syncytiotrophoblast . autophagy has roles not only in starvation response , but also in keeping quality of protein 
 and organelle , development , differentiation , antigen presentation , tumorigenesis , senescence 
 and more [ 10 , 21 , 25 , 26 , 30 , 32 , 33 , 38 ] . additionally , as human atg8 homolog , there are gabarap , gabarapl1 and 
 gabarapl2 besides lc3 family [ 19 , 28 , 31 , 35 ] . considering from a great deal of variety , it is not 
 strange that these are participating in difference of autophagy . in this study , we observed 
 only lc3a , b and c. gabarap family may have roles for other function and/or pathway and 
 redundant roles . to clarify all varieties of autophagy , further researches will be 
 required . in conclusion , the present study strongly suggests that autophagy could be related to 
 placental invasion system and that lc3b is the most sensitive to nutrient starvation in the 
 lc3 family .
lc3  the mammalian homolog of atg8  was found as autophagosome membrane binding protein 
 in mammals and widely used as an autophagosomal marker . 
 lc3a , b and c show different 
 expression patterns in each tissue . 
 the aim of this study was to reveal the differences of 
 expression patterns among lc3 families in mouse placenta under normal condition and 
 nutrient starving condition . 
 lc3a and b were highly expressed in decidual cells . 
 lc3a and 
 b were increased in d14 compared with d12 and d16 in mouse placenta , while lc3c was 
 decreased . 
 starvation induced increase in lc3b expression specifically . 
 immunohistochemistry showed different expression patterns among lc3a , b and c. lc3a 
 expression in syncytiotrophoblast was vanished by starvation . 
 the results of real time 
 rt - pcr suggested differences between d12 and d16 in autophagic cascade induced by 
 starvation . taken together , this study suggests that autophagy could play a role in 
 placental invasion system and that nutrient starvation affects lc3b expression .