the data has been deposited with ncbi 's geo database as record gse57496 , http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse57496 . the gingival tissue used was attached gingiva obtained from healthy , young ( mean age : 2535  years ; five males , four females ) non - smoking patients of caucasian descent . the study conformed to the declaration of helsinki guidelines and was approved by the creighton university institutional review board . gf were isolated by rinsing the gingival tissue six times [ in hank 's balanced salt solution ( hbss ) , invitrogen , grand island , ny , usa ] , in order to diminish microbial contaminants and then finely mincing with scalpels inside a 35  mm dish ( falcon , fisher scientific , waltham , ma , usa ) containing 2  ml of complete media [ dulbecco 's modified eagle medium ( dmem , invitrogen ) , with 10% defined fetal bovine serum ( hyclone , logan , ut , usa ) and 100  g / ml primocin ( invivogen , san diego , ca , usa ) ] . the gf were allowed to explant from the minced tissue ( mean time ~  14  days ) in complete media with changes every three days . of note , the gf isolates we have used to study mitogenesis , growth , chemotaxis , in vitro wound - healing , and elevation of intracellular calcium , , ,   were cultured from young , healthy male and female patients in the same age range as the donors for this study . statistical analyses showed no detectable response differences to lpa 18:1 treatment between isolates . in order to preserve cellular responses closest to in vivo , pass 2 gf the gf were seeded at 1    10 cells/2  ml in 35  mm dishes , and incubated in a 5% co2 , 37  c cell culture incubator for two days until approximately 90% confluent . the cells were washed four times with hbss and then serum - starved for 24  h in serum - free dmem with primocin . this step was done to make sure that any lpa - mediated effects from the serum the cells were cultured in would be significantly diminished or absent . human gf can be completely deprived of serum for this period of time ( unpublished observations from our previous studies ) without triggering apoptosis . louis , mo , usa ) stock was prepared at 1    10  m in 0.25% de - lipidated ( fraction v ) bovine serum albumin ( bsa ) ( sigma - aldrich ) in serum - free dmem . this is one of the major unsaturated lpa species ( 1,2 ) that is present in normal human saliva ( 3,8 ) . each donor 's cells were individually treated for 2  h or 8  h with 2  ml 1    10  m lpa in serum - free dmem . controls were treated with an equal volume of 0.25% fraction v bsa in serum - free dmem . the cells from each donor were individually trypsinized , washed once with ice - cold phosphate - buffered saline ( pbs ) , held on wet ice , and counted . the cells from the nine donors were then grouped into 3 pools of 3000 cells in total , each containing 1000 cells / donor [ pool a  =  donors 13 ( 2 m , 1 f ) , pool b  =  donors 46 ( 1 m , 2 f ) , and pool c  =  donors 79 ( 2 m , 1 f ) ] . miltenyi 's biotec 's ( san diego , ca , usa ) proprietary superamp buffer was used to directly lyse the cell pools using their protocol for cells ( http://www.miltenyibiotec.com/~/media/images/products/import/0006000/im0006042.ashx ) . the lysates were quick - frozen on dry ice and shipped to the company for microarray analysis . as we avoided larger - scale expansion of the pass 2 gf isolates beyond 35  mm dishes , the superamp technology was then utilized at miltenyi biotec . their proprietary amplification method is based on a global pcr protocol using cdna derived from mrna . magnetic bead ( macs ) technology was used to isolate the mrna . the average length of the highly amplified cdna products ranged between 2001000  bp . a nd-1000 spectrophotometer ( nanodrop technologies , wilmington , de , usa ) was used to quantify the cdna . the integrity of the cdna was checked via the agilent 2100 bioanalyzer platform ( agilent technologies , santa clara , ca , usa ) . lpa - induced gene transcription for each treatment pool and controls was assessed by the company . cdnas ( 250  ng of each sample ) were used as template for cy3 and cy5 labeling , which was performed according to miltenyi 's proprietary protocol . the corresponding cy3- and cy5-labeled cdnas were combined and hybridized overnight ( 17  h , 65  c ) to an agilent whole human genome oligo microarrays 4    44  k using agilent 's recommended hybridization chamber and oven . control samples were labeled with cy3 and experimental samples were labeled with cy5 . in the final steps , the microarrays were washed once for 1  min at room temperature with 6 sspe buffer containing 0.005% n - lauroylsarcosine , followed by a second  1  min wash with pre - heated ( 37  c ) 0.06 sspe buffer containing 0.005% n - lauroylsarcosine . the last washing step ( 30  s ) was performed with acetonitrile . the gingival tissue used was attached gingiva obtained from healthy , young ( mean age : 2535  years ; five males , four females ) non - smoking patients of caucasian descent . the study conformed to the declaration of helsinki guidelines and was approved by the creighton university institutional review board . gf were isolated by rinsing the gingival tissue six times [ in hank 's balanced salt solution ( hbss ) , invitrogen , grand island , ny , usa ] , in order to diminish microbial contaminants and then finely mincing with scalpels inside a 35  mm dish ( falcon , fisher scientific , waltham , ma , usa ) containing 2  ml of complete media [ dulbecco 's modified eagle medium ( dmem , invitrogen ) , with 10% defined fetal bovine serum ( hyclone , logan , ut , usa ) and 100  g / ml primocin ( invivogen , san diego , ca , usa ) ] . the gf were allowed to explant from the minced tissue ( mean time ~  14  days ) in complete media with changes every three days . of note , the gf isolates we have used to study mitogenesis , growth , chemotaxis , in vitro wound - healing , and elevation of intracellular calcium , , ,   were cultured from young , healthy male and female patients in the same age range as the donors for this study . in order to preserve cellular responses closest to in vivo , pass 2 gf were used for mrna isolation . the gf were seeded at 1    10 cells/2  ml in 35  mm dishes , and incubated in a 5% co2 , 37  c cell culture incubator for two days until approximately 90% confluent . the cells were washed four times with hbss and then serum - starved for 24  h in serum - free dmem with primocin . this step was done to make sure that any lpa - mediated effects from the serum the cells were cultured in would be significantly diminished or absent . human gf can be completely deprived of serum for this period of time ( unpublished observations from our previous studies ) without triggering apoptosis . louis , mo , usa ) stock was prepared at 1    10  m in 0.25% de - lipidated ( fraction v ) bovine serum albumin ( bsa ) ( sigma - aldrich ) in serum - free dmem . this is one of the major unsaturated lpa species ( 1,2 ) that is present in normal human saliva ( 3,8 ) . each donor 's cells were individually treated for 2  h or 8  h with 2  ml 1    10  m lpa in serum - free dmem . controls were treated with an equal volume of 0.25% fraction v bsa in serum - free dmem . the cells from each donor were individually trypsinized , washed once with ice - cold phosphate - buffered saline ( pbs ) , held on wet ice , and counted . the cells from the nine donors were then grouped into 3 pools of 3000 cells in total , each containing 1000 cells / donor [ pool a  =  donors 13 ( 2 m , 1 f ) , pool b  =  donors 46 ( 1 m , 2 f ) , and pool c  =  donors 79 ( 2 m , 1 f ) ] . miltenyi 's biotec 's ( san diego , ca , usa ) proprietary superamp buffer was used to directly lyse the cell pools using their protocol for cells ( http://www.miltenyibiotec.com/~/media/images/products/import/0006000/im0006042.ashx ) . the lysates were quick - frozen on dry ice and shipped to the company for microarray analysis . as we avoided larger - scale expansion of the pass 2 gf isolates beyond 35  mm dishes , the superamp technology was then utilized at miltenyi biotec . their proprietary amplification method is based on a global pcr protocol using cdna derived from mrna . the average length of the highly amplified cdna products ranged between 2001000  bp . a nd-1000 spectrophotometer ( nanodrop technologies , wilmington , de , usa ) was used to quantify the cdna . the integrity of the cdna was checked via the agilent 2100 bioanalyzer platform ( agilent technologies , santa clara , ca , usa ) . lpa - induced gene transcription for each treatment pool and controls was assessed by the company . cdnas ( 250  ng of each sample ) were used as template for cy3 and cy5 labeling , which was performed according to miltenyi 's proprietary protocol . the corresponding cy3- and cy5-labeled cdnas were combined and hybridized overnight ( 17  h , 65  c ) to an agilent whole human genome oligo microarrays 4    44  k using agilent 's recommended hybridization chamber and oven . control samples were labeled with cy3 and experimental samples were labeled with cy5 . in the final steps , the microarrays were washed once for 1  min at room temperature with 6 sspe buffer containing 0.005% n - lauroylsarcosine , followed by a second  1  min wash with pre - heated ( 37  c ) 0.06 sspe buffer containing 0.005% n - lauroylsarcosine . the microarray image files were read out and processed using agilent feature extraction software ( fes ) , which reads feature intensities and ratios ( including background subtraction and normalization ) , rejects outliers and calculates statistical confidences ( p - values ) . the output data of the agilent feature extraction software includes gene lists with the complete raw data sets , referred to as single - experiment raw data list ; the complete descriptions can be found in the agilent g2567aa feature . for determination of differential gene expression fes - derived output data files , miltenyi further analyzed the data using the rosetta resolver gene expression data analysis system ( rosetta biosoftware , www.rosettabio.com ) . this software 's analytical range includes the capacity to visualize the data analysis results as a double - log scatter plot . a double - log scatter plot for experiment number one is shown in fig .  2 as an example . rosetta resolver analysis results in a level of significance of p    0.01 . thus , genes from the microarray experiments which showed  , or  2-fold differences between the signal from the lpa - treated samples and their untreated controls were designated as significantly changed .
the pleiotropic , bioactive lipid lysophosphatidic acid [ ( lpa ) , 1-acyl - sn - glycerol-3-phosphate ] exerts critical regulatory actions in physiology and pathophysiology in many systems . 
 it is present in normal bodily fluids , and is elevated in pathology ( 1 ) . in vivo , 
 lpa  exists as distinct molecular species , each having a single fatty acid of varying chain length and degree of unsaturation covalently attached to the glycerol backbone via an acyl , alkyl , or alkenyl link . 
 these species differ in affinities for the individual lpa receptors [ ( lpars ) , lpa1 - 6 ] and coupling to g proteins ( 2 ) . 
 however , lpa 18:1 has been and continues to be the most commonly utilized species in reported studies . 
 the actions of  lpa  remain poorly defined in oral biology and pathophysiology . 
 our laboratory has addressed this knowledge gap by studying in vitro the actions of the major human salivary lpa species [ 18:1 , 18:0 , and 16:0 ( 3 ) ] in human oral cells [ 4 ] , [ 5 ] , [ 6 ] , [ 7 ] . 
 this includes gingival fibroblasts ( gf ) , which our flow cytometry data from multiple donors found that they express lpa1 - 5 ( 6 ) . 
 we have also reported that these species are ten - fold elevated to pharmacologic levels in the saliva and gingival crevicular fluid obtained from patients with moderate 
 severe periodontitis ( 8) . as the potential of lpa to regulate transcriptional activity had not been examined in the oral system , this study used whole human genome microarray analysis to test the hypothesis that lpa 18:1-treated human gf would show significant changes in gene transcripts relevant to their biology , wound - healing , and inflammatory responses . 
 lpa 18:1 was found to significantly regulate a large , complex set of genes critical to gf biology in these categories and to periodontal disease . 
 the raw data has been deposited at ncbi 's geo database as record gse57496 .