mouse luecs were immortalized with polyoma middle t antigen and cultured in mcdb131 medium ( gibco ; life technologies ) supplemented with 20% fbs ( invitrogen ) , 2  mm  l - glutamine ( lonza ) , 1  mm na - pyruvate ( gibco ; life technologies ) , 100  g / ml heparin ( sigma - aldrich ) , and 50  g / ml ec growth supplement ( ecgs ) obtained from calf brain .
ecs were seeded on plates coated with glutaraldehyde - crosslinked gelatin and cultured in complete medium for 4  days to reach confluence .
total rna was extracted from ~  5    10 cells using the rneasy mini kit ( qiagen ) following manufacturer 's instruction .
quality control of the rna samples was performed using an agilent bioanalyzer 2100 ( agilent technologies ) .
the rna from three independent extractions was processed for each of the cell line under analysis .
a total of 150  ng of rna from each sample was used for rna quality check , labeling and hybridization on a mouse gene 1.0 st genechip array according to the manufacturer 's instructions ( affymetrix ) .
three independent biological replicates were performed for each condition ( l1-transfected vs. mock - transfected luecs ) .
we used the robust multi - array average ( rma )   to normalized data .
a total of 35,512 probesets were loaded and 819,041  pm probes were used for analysis .
next , we uploaded normalized data in brb arraytools ( http://linus.nci.nih.gov/brb-arraytools.html ) to run sam analysis ( http://www-stat.stanford.edu/~tibs/sam/ ) in order to identify differentially expressed genes upon l1 overexpression in luec cells
. the sam parameters of analysis were the following : number of classes : 2number of probesets : 35,512target proportion of false discoveries ( q - value ) : 0.05delta value used to identify significant probesets : 1.24701fudge factor for standard deviation computed : 0.04825 .
number of probesets : 35,512 target proportion of false discoveries ( q - value ) : 0.05 delta value used to identify significant probesets : 1.24701 fudge factor for standard deviation computed : 0.04825 . under these conditions
, we identified a total of 3409 significant probesets corresponding to 2684 unique annotated genes ( fig .  1 ) .
we then selected probesets having   1.5 fold change difference ( l1 vs. ctr ) that resulted in a set of 496 upregulated and 743 downregulated probesets annotated , corresponding to 361 upregulated and 580 downregulated unique genes , referred to as the 
l1-ecs signature. next , we uploaded the l1-ecs signature ( probeset level ) in the ingenuity pathway analysis software ( ipa , http://www.ingenuity.com ) to identify biological functions / pathways putatively regulated by l1 .
the mouse gene 1.0 st array reference set present in the ipa database was used for the  ipa core analysis  .
only direct relationships in mammals ( i.e. , human , mouse , and rat ) were considered , including endogenous chemicals for gene network analysis .
the p - values for biofunction enrichment were corrected for multiple testing using the benjamini - hochberg correction . with these settings we identified 23 bio - functions significantly enriched ( p - value  <  0.05 ; benjiamini - hochberg correction ) in l1-overexpressing cells compared to mock - transfected cells . considering the massive changes in transcriptional activity and the predicted effect on a variety of biological functions upon l1 overexpression , we then asked whether l1 could activate / inhibit transcription factor(s ) ( tfs ) . to address this question
such an analysis allows the identification of transcriptional regulators activated / inhibited under specific experimental conditions , thus accounting for the observed gene expression changes .
strikingly , we identified a total of 18 and 11 tfs predicted to be activated or inhibited by l1 , respectively . of note , 5 tfs were differentially regulated in l1-overexpressing vs. mock - transfected cells and , in particular , four of the activated tfs were upregulated in l1 overexpressing cells ( stat1 , stat2 , irf7 and atf4 ) , while one of the inhibited tfs was downregulated ( foxm1 ) .
we then performed the mechanistic networks analysis to further explore the contribution of the tfs in regulating gene networks .
we found that stat1 , stat2 , irf7 , atf4 and stat3 ( that was among the 18 tfs predicted to be activated ) interact with each other ( fig . 
2 ) , thus exerting a coordinated control on a directional network of 105 genes regulated by l1 ( i.e. 11% of all the l1-regulated genes ) .
most of these genes were consistently up- or downregulated with the expression change of their upstream tfs .
these findings were supported by functional studies which implicated the jak / stat signaling pathway in the biological response of luecs to l1 overexpression .
mouse luecs were immortalized with polyoma middle t antigen and cultured in mcdb131 medium ( gibco ; life technologies ) supplemented with 20% fbs ( invitrogen ) , 2  mm  l - glutamine ( lonza ) , 1  mm na - pyruvate ( gibco ; life technologies ) , 100  g / ml heparin ( sigma - aldrich ) , and 50  g / ml ec growth supplement ( ecgs ) obtained from calf brain .
ecs were seeded on plates coated with glutaraldehyde - crosslinked gelatin and cultured in complete medium for 4  days to reach confluence .
total rna was extracted from ~  5    10 cells using the rneasy mini kit ( qiagen ) following manufacturer 's instruction .
quality control of the rna samples was performed using an agilent bioanalyzer 2100 ( agilent technologies ) .
the rna from three independent extractions was processed for each of the cell line under analysis .
a total of 150  ng of rna from each sample was used for rna quality check , labeling and hybridization on a mouse gene 1.0 st genechip array according to the manufacturer 's instructions ( affymetrix ) .
three independent biological replicates were performed for each condition ( l1-transfected vs. mock - transfected luecs ) .
we used the robust multi - array average ( rma )   to normalized data .
a total of 35,512 probesets were loaded and 819,041  pm probes were used for analysis .
next , we uploaded normalized data in brb arraytools ( http://linus.nci.nih.gov/brb-arraytools.html ) to run sam analysis ( http://www-stat.stanford.edu/~tibs/sam/ ) in order to identify differentially expressed genes upon l1 overexpression in luec cells
. the sam parameters of analysis were the following : number of classes : 2number of probesets : 35,512target proportion of false discoveries ( q - value ) : 0.05delta value used to identify significant probesets : 1.24701fudge factor for standard deviation computed : 0.04825 .
number of probesets : 35,512 target proportion of false discoveries ( q - value ) : 0.05 delta value used to identify significant probesets : 1.24701 fudge factor for standard deviation computed : 0.04825 . under these conditions
, we identified a total of 3409 significant probesets corresponding to 2684 unique annotated genes ( fig .  1 ) .
we then selected probesets having   1.5 fold change difference ( l1 vs. ctr ) that resulted in a set of 496 upregulated and 743 downregulated probesets annotated , corresponding to 361 upregulated and 580 downregulated unique genes , referred to as the 
l1-ecs signature. next , we uploaded the l1-ecs signature ( probeset level ) in the ingenuity pathway analysis software ( ipa , http://www.ingenuity.com ) to identify biological functions / pathways putatively regulated by l1 .
the mouse gene 1.0 st array reference set present in the ipa database was used for the  ipa core analysis  . only direct relationships in mammals ( i.e. , human , mouse , and rat )
the p - values for biofunction enrichment were corrected for multiple testing using the benjamini - hochberg correction . with these settings we identified 23 bio - functions significantly enriched ( p - value  <  0.05 ; benjiamini - hochberg correction ) in l1-overexpressing cells compared to mock - transfected cells . considering the massive changes in transcriptional activity and the predicted effect on a variety of biological functions upon l1 overexpression
, we then asked whether l1 could activate / inhibit transcription factor(s ) ( tfs ) . to address this question
such an analysis allows the identification of transcriptional regulators activated / inhibited under specific experimental conditions , thus accounting for the observed gene expression changes .
strikingly , we identified a total of 18 and 11 tfs predicted to be activated or inhibited by l1 , respectively . of note ,
5 tfs were differentially regulated in l1-overexpressing vs. mock - transfected cells and , in particular , four of the activated tfs were upregulated in l1 overexpressing cells ( stat1 , stat2 , irf7 and atf4 ) , while one of the inhibited tfs was downregulated ( foxm1 ) .
we then performed the mechanistic networks analysis to further explore the contribution of the tfs in regulating gene networks .
we found that stat1 , stat2 , irf7 , atf4 and stat3 ( that was among the 18 tfs predicted to be activated ) interact with each other ( fig . 
2 ) , thus exerting a coordinated control on a directional network of 105 genes regulated by l1 ( i.e. 11% of all the l1-regulated genes ) .
most of these genes were consistently up- or downregulated with the expression change of their upstream tfs .
these findings were supported by functional studies which implicated the jak / stat signaling pathway in the biological response of luecs to l1 overexpression .
here we describe the methods of analysis that were used to define and characterize the gene expression profile induced by l1 in murine lung endothelial cells ( luecs ) .
we used the affymetrix mouse gene 1.0 st arrays , which enabled us to produce gene expression data of a total of 28,869 genes ( 764,885 distinct probes ) . through the combined use of the significant analysis of microarrays ( sam ) and the ingenuity pathway analysis ( ipa )
, we identified a set of 941 genes and 23 bio - functions significantly regulated in l1-overexpressing vs. control luecs .
we reasoned that these massive transcriptional changes could be the result of the l1-mediated regulation of tfs .
indeed , by using the  upstream modulator analysis  in ipa we found that 29 tfs were predicted to be activated / inhibited and , among these , 5 tfs were transcriptionally regulated in our profiling experiment . among the 29 tfs identified ,
many are involved in the inflammatory and/or angiogenic cascades ( stat1 , stat2 , stat3 , irf1 , irf3 , irf7 , rela / p65 , nfkb1 and atf4 ) . of note , while l1 has been causally linked to nf-b signaling in cancer cells , we are the first to report a crosstalk between l1 and stats , irfs and atf4 . our data , therefore , point to l1 as a novel orchestrator of the cancer - associated response that entails the regulation of gene networks and biochemical cascades which , in turn , impact on the pathophysiology of cancer cells as well as tumor microenvironment . importantly ,
the results of our in silico analysis have been successfully validated experimentally , thus lending further support to their biological relevance .
 we recently identified a novel role for the l1 transmembrane glycoprotein ( also known as l1cam or cd171 ) in the regulation of tumor angiogenesis and vessels stabilization . 
 l1 overexpression in cultured endothelial cells of the lung ( luecs ) exerted a pleiotropic effect in that it regulated proliferation , migration , tubulogenesis , vascular permeability , and endothelial - to - mesenchymal transition ( endmt ) . 
 in addition , we provided strong evidence that antibody - mediated targeting of l1 may be an effective strategy for vessel normalization with the potential to increase efficacy of chemotherapeutic agents.high-throughput microarray expression profile revealed that l1 modulates the expression of hundreds of genes mainly involved in cell cycle regulation , dna replication , cellular assembly , migration , development and organization . by using a  pathway - oriented analysis strategy 
 we were able to identify a network of 105 genes modulated by l1 through the predicted activation of five transcription factors : stat1 , stat2 , stat3 , irf7 , and atf4 . 
 indeed , l1 overexpression resulted in the strong induction of stat3 phosphorylation which was abolished by antibody - mediated neutralization of il-6r. these results indicated that l1 promoted stat3 activation via the il-6/il-6r axis . 
Direct link to deposited data
Experimental design, materials and methods
Cell culture
RNA preparation
Gene expression profiling
Discussion
Conflict of interest