tissue was collected from postnatal - day-21 albino wistar rats ( rattus norvegivus ) that were euthanized by asphyxiation and subsequent cervical dislocation . all procedures were approved by the animal ethical review committee of the university of sydney , australia and were carried out in accordance with the national health medical research council ( australia ) guidelines and the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research ( united states ) . lens epithelial explants were prepared as described previously and cultured in medium 199 with earle 's salts ( life technologies , waltham , ma , usa ) , supplemented with 50 g / ml l - glutamine , 50 iu / ml penicillin/50 g / ml streptomycin ( thermo fisher scientific , waltham , ma , usa ) , 2.5 g / ml amphostat b ( thermo fisher scientific ) , and 0.1% bsa ( sigma - aldrich corp . , recombinant tgf-2 was added to media in each culture dish at a working concentration ranging from 200 pg / ml up to 1ng / ml ( r&d systems , minneapolis , mn , usa ) . before the addition of tgf-2 , some explants were pretreated with the pan - nadph oxidase inhibitor vas2870 at a working concentration of 5 m . the progression of emt in live cells was observed and captured using phase - contrast microscopy ( olympus ck2 , tokyo , japan ) and a digital camera ( leica dfc-280 ; leica camera , wetzlar , germany ) . percentage cell loss quantification was performed using the thresholding function of imagej ( national institutes of health [ nih ] , bethesda , md , usa ) , such that cells could be distinguished from bare lens capsule . at the end of the culture period , explants were fixed in 100% methanol for 45 seconds , followed by 3 consecutive 15-second rinses in pbs . explants were blocked in 10% normal goat serum ( ngs ) for 1 hour at room temperature . excess normal goat serum was removed and primary antibody was applied , diluted in 0.15% ngs / pbs supplemented with 1% bsa . -smooth muscle actin ( sma ) was labeled with a specific monoclonal mouse antibody ( sigma - aldrich corp . ) , diluted 1:100 . nox4 was labeled with a specific polyclonal rabbit antibody ( santa cruz biotechnology , dallas , tx , usa ) , diluted 1:50 . e - cadherin was labeled with a specific polyclonal mouse antibody ( bd biosciences , north ryde , nsw , australia ) , diluted at 1:100 . the following day , dishes were equilibrated to room temperature and subject to 3  5 minute washes in pbs / bsa . an appropriate secondary antibody was diluted in pbs / bsa and applied to each explant for 2 hours in dark conditions . -smooth muscle actin and e - cadherin were detected using goat anti - mouse alexa - fluor 488 ( cell signaling , danvers , ma , usa ) . nox4 was detected using an anti - rabbit whole igg conjugated to cy3 ( sigma - aldrich corp . ) . dishes subsequently were rinsed in pbs / bsa and a 1:2000 solution of bisbenzimide ( hoechst dye ) diluted in pbs / bsa was applied for 3 minutes to visualize cell nuclei . immunofluorescent labeling was viewed and captured using epifluorescence microscopy ( leica - dmlb ; leica camera ) , and a digital camera ( micropublisher 3.3 rtv ; q - imaging , surrey , bc canada ) . where appropriate , fluorescence also was visualized and images were collected using a zeiss lsm-5pa confocal microscope ( carl zeiss ag , jena , germany ) . after appropriate treatment , lens epithelial explants were placed into a 1.5 ml centrifuge tube . total rna extraction was performed using a quick - rna miniprep kit ( zymo research , irvine , ca , usa ) according to manufacturer 's instructions . first - strand cdna synthesis was done using 150 ng of rna with a reverse transcription system ( bioline , eveleig , nsw , australia ) according to the manufacturer 's instructions . control reactions omitting reverse transcriptase were set up and later tested , confirming that genomic dna was absent from the purified rna template ( data not shown ) . for pcr amplification , the mytaq red dna polymerase ( bioline ) kit primers used for the detection of nox14 and gapdh mrna were based on those previously published . for detection of nox14 mrna , sequence - specific primers were used : nox1 : fw : 5-cttcctcactggctgggata-3 , rev : 5-tgacagcatttgcgcaggc-3 ; nox2 : 5-ccagtgaagatgtgttcagct-3 , 5-gcacagccagtagaagtagat-3 ; nox3 : 5-gtgtccttcgtggttgctct-3 , 5-tctacagcacacgctcgttc-3 ; nox4 : 5-agtcaaacagatggga-3 , 5-tgtcccatatgagttgtt-3 ; gapdh : 5-tgaacgggaagctcactgg-3 , 5-tccaccaccctgttgctgta-3. cdna was amplified for 35 cycles : nox1 and nox2 : 95c for 45 seconds , 51c for 45 seconds , and 72c for 45 seconds ; nox1 , nox3 , and gapdh : 95c for 30 seconds , 62c for 30 seconds , and 72c for 30 seconds . amplified dna was visualized with sybr safe dna gel stain ( diluted at 1:10,000 ; invitrogen , carlsbad , ca , usa ) in a 2% agarose tae gel , run at 100 v for 20 minutes . positive control tissues for each nox homologue also were examined ( nox1 , rat colon ; nox2 and nox4 , rat kidney ) . dihydroethidium ( life technologies ) was added to explants in each dish containing 1 ml of medium to yield a working concentration of 30 m . explants then were rinsed in cold pbs ( 3  15 seconds ) before being mounted in 10% glycerol / pbs . in its reduced form , dhe fluoresces blue , but when exposed to ros , including the superoxide anion radical and hydrogen peroxide , it readily reacts to form the oxidized red fluorescent dhe form that can be viewed readily using epifluorescence microscopy . fluorescence microscopy gives an indication of the level of total ros produced by tgf-2dependent nox4 ; however , absolute quantification of specific types of ros can not be determined using this method . after appropriate treatment , lens epithelial explants were placed into a 1.5 ml tube with cold lysis buffer containing 2.5 mm edta , 25 mm tris , 0.375 m nacl , 250 mm sodium orthovandate , 10 mm sodium deoxycholate , and a protease inhibitor cocktail ( complete , mini ; roche , basel , switzerland ) . tubes were rotated for 2 hours and protein lysates were extracted by centrifuging for 15 minutes ( 15,500 g , 4c ) . the direct detect spectrophotometer system ( merck millipore , bayswater , vic , australia ) was used to determine the protein concentration for each lysate , according to manufacturer 's instructions . equal amounts of protein per sample were loaded onto 10% sds - page gels for electrophoresis at 200 v for 1.5 hours and transferred onto a polyvinylidene fluoride membrane ( merck millipore ) at 100 v for 1 hour . membranes then were incubated with a blocking solution containing 5% skim milk diluted in 0.1% tween-20 in tris - buffered saline ( tbs - t ) for 1 hour at room temperature and probed overnight at 4c with specific antibodies , appropriately diluted in blocking buffer . rabbit monoclonal anti - nox4 antibody ( santa - cruz biotechnology ) was diluted at 1:200 , mouse monoclonal anti--sma ( sigma - aldrich corp . ) was diluted at 1:2000 and mouse monoclonal anti - gapdh ( sigma - aldrich corp . ) membranes were then rinsed ( 3  5 minutes ) in tbs - t and anti - rabbit or anti - mouse secondary antibodies conjugated to horseradish peroxidase ( diluted at 1:5000 in tbs - t ) were applied to each membrane and incubated at room temperature for 2 hours with gentle rocking . membranes were rinsed ( 3  5 minutes ) with tbs - t and an equal volume of peroxidase and luminol reagent was applied immediately before imaging using the chemidoc mp imaging system ( bio - rad laboratory , hercules , ca , usa ) . tissue was collected from postnatal - day-21 albino wistar rats ( rattus norvegivus ) that were euthanized by asphyxiation and subsequent cervical dislocation . all procedures were approved by the animal ethical review committee of the university of sydney , australia and were carried out in accordance with the national health medical research council ( australia ) guidelines and the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research ( united states ) . lens epithelial explants were prepared as described previously and cultured in medium 199 with earle 's salts ( life technologies , waltham , ma , usa ) , supplemented with 50 g / ml l - glutamine , 50 iu / ml penicillin/50 g / ml streptomycin ( thermo fisher scientific , waltham , ma , usa ) , 2.5 g / ml amphostat b ( thermo fisher scientific ) , and 0.1% bsa ( sigma - aldrich corp . , st . recombinant tgf-2 was added to media in each culture dish at a working concentration ranging from 200 pg / ml up to 1ng / ml ( r&d systems , minneapolis , mn , usa ) . before the addition of tgf-2 , some explants were pretreated with the pan - nadph oxidase inhibitor vas2870 at a working concentration of 5 m . the progression of emt in live cells was observed and captured using phase - contrast microscopy ( olympus ck2 , tokyo , japan ) and a digital camera ( leica dfc-280 ; leica camera , wetzlar , germany ) . percentage cell loss quantification was performed using the thresholding function of imagej ( national institutes of health [ nih ] , bethesda , md , usa ) , such that cells could be distinguished from bare lens capsule . at the end of the culture period , explants were fixed in 100% methanol for 45 seconds , followed by 3 consecutive 15-second rinses in pbs . explants were blocked in 10% normal goat serum ( ngs ) for 1 hour at room temperature . excess normal goat serum was removed and primary antibody was applied , diluted in 0.15% ngs / pbs supplemented with 1% bsa . -smooth muscle actin ( sma ) was labeled with a specific monoclonal mouse antibody ( sigma - aldrich corp . ) , diluted 1:100 . nox4 was labeled with a specific polyclonal rabbit antibody ( santa cruz biotechnology , dallas , tx , usa ) , diluted 1:50 . e - cadherin was labeled with a specific polyclonal mouse antibody ( bd biosciences , north ryde , nsw , australia ) , diluted at 1:100 . the following day , dishes were equilibrated to room temperature and subject to 3  5 minute washes in pbs / bsa . an appropriate secondary antibody was diluted in pbs / bsa and applied to each explant for 2 hours in dark conditions . -smooth muscle actin and e - cadherin were detected using goat anti - mouse alexa - fluor 488 ( cell signaling , danvers , ma , usa ) . nox4 was detected using an anti - rabbit whole igg conjugated to cy3 ( sigma - aldrich corp . ) . dishes subsequently were rinsed in pbs / bsa and a 1:2000 solution of bisbenzimide ( hoechst dye ) diluted in pbs / bsa was applied for 3 minutes to visualize cell nuclei . immunofluorescent labeling was viewed and captured using epifluorescence microscopy ( leica - dmlb ; leica camera ) , and a digital camera ( micropublisher 3.3 rtv ; q - imaging , surrey , bc canada ) . where appropriate , fluorescence also was visualized and images were collected using a zeiss lsm-5pa confocal microscope ( carl zeiss ag , jena , germany ) . after appropriate treatment , lens epithelial explants were placed into a 1.5 ml centrifuge tube . total rna extraction was performed using a quick - rna miniprep kit ( zymo research , irvine , ca , usa ) according to manufacturer 's instructions . first - strand cdna synthesis was done using 150 ng of rna with a reverse transcription system ( bioline , eveleig , nsw , australia ) according to the manufacturer 's instructions . control reactions omitting reverse transcriptase were set up and later tested , confirming that genomic dna was absent from the purified rna template ( data not shown ) . for pcr amplification , the mytaq red dna polymerase ( bioline ) kit primers used for the detection of nox14 and gapdh mrna were based on those previously published . for detection of nox14 mrna , sequence - specific primers were used : nox1 : fw : 5-cttcctcactggctgggata-3 , rev : 5-tgacagcatttgcgcaggc-3 ; nox2 : 5-ccagtgaagatgtgttcagct-3 , 5-gcacagccagtagaagtagat-3 ; nox3 : 5-gtgtccttcgtggttgctct-3 , 5-tctacagcacacgctcgttc-3 ; nox4 : 5-agtcaaacagatggga-3 , 5-tgtcccatatgagttgtt-3 ; gapdh : 5-tgaacgggaagctcactgg-3 , 5-tccaccaccctgttgctgta-3. cdna was amplified for 35 cycles : nox1 and nox2 : 95c for 45 seconds , 51c for 45 seconds , and 72c for 45 seconds ; nox1 , nox3 , and gapdh : 95c for 30 seconds , 62c for 30 seconds , and 72c for 30 seconds . amplified dna was visualized with sybr safe dna gel stain ( diluted at 1:10,000 ; invitrogen , carlsbad , ca , usa ) in a 2% agarose tae gel , run at 100 v for 20 minutes . positive control tissues for each nox homologue also were examined ( nox1 , rat colon ; nox2 and nox4 , rat kidney ) . dihydroethidium ( life technologies ) was added to explants in each dish containing 1 ml of medium to yield a working concentration of 30 m . explants then were rinsed in cold pbs ( 3  15 seconds ) before being mounted in 10% glycerol / pbs . in its reduced form , dhe fluoresces blue , but when exposed to ros , including the superoxide anion radical and hydrogen peroxide , it readily reacts to form the oxidized red fluorescent dhe form that can be viewed readily using epifluorescence microscopy . fluorescence microscopy gives an indication of the level of total ros produced by tgf-2dependent nox4 ; however , absolute quantification of specific types of ros can not be determined using this method . after appropriate treatment , lens epithelial explants were placed into a 1.5 ml tube with cold lysis buffer containing 2.5 mm edta , 25 mm tris , 0.375 m nacl , 250 mm sodium orthovandate , 10 mm sodium deoxycholate , and a protease inhibitor cocktail ( complete , mini ; roche , basel , switzerland ) . tubes were rotated for 2 hours and protein lysates were extracted by centrifuging for 15 minutes ( 15,500 g , 4c ) . the direct detect spectrophotometer system ( merck millipore , bayswater , vic , australia ) was used to determine the protein concentration for each lysate , according to manufacturer 's instructions . equal amounts of protein per sample were loaded onto 10% sds - page gels for electrophoresis at 200 v for 1.5 hours and transferred onto a polyvinylidene fluoride membrane ( merck millipore ) at 100 v for 1 hour . membranes then were incubated with a blocking solution containing 5% skim milk diluted in 0.1% tween-20 in tris - buffered saline ( tbs - t ) for 1 hour at room temperature and probed overnight at 4c with specific antibodies , appropriately diluted in blocking buffer . rabbit monoclonal anti - nox4 antibody ( santa - cruz biotechnology ) was diluted at 1:200 , mouse monoclonal anti--sma ( sigma - aldrich corp . ) was diluted at 1:2000 and mouse monoclonal anti - gapdh ( sigma - aldrich corp . ) membranes were then rinsed ( 3  5 minutes ) in tbs - t and anti - rabbit or anti - mouse secondary antibodies conjugated to horseradish peroxidase ( diluted at 1:5000 in tbs - t ) were applied to each membrane and incubated at room temperature for 2 hours with gentle rocking . membranes were rinsed ( 3  5 minutes ) with tbs - t and an equal volume of peroxidase and luminol reagent was applied immediately before imaging using the chemidoc mp imaging system ( bio - rad laboratory , hercules , ca , usa ) . nox4 expression in tgf-driven lens emt was assayed using immunolabeling of lens epithelial explant lysates . specifically , we determined nox4 expression using western blotting at different time points ( 0 , 2 , 4 , 6 , and 8 hours ) . 1 ) , and this level was significantly different to that of the untreated control at the same time period ( fig . expression of nox4 also was significantly more pronounced at 8 hours to that observed at 6 hours ( fig . 8 hours after tgf-2 treatment , the level of nox4 protein expression was approximately 2.5-fold higher to that of the matching control without tgf-2 ( fig . this increase in nox4 protein expression also was accompanied by an upregulation in nox4 mrna transcription in tgf-2treated explants compared to untreated explants at 8 hours ( fig . transforming growth factor-2 induces an increase in nox4 expression in lens epithelial explants : western blot . ( a ) representative western blot depicting the time - course ( 4 , 6 , and 8 hours ) of nox4 expression following tgf-2 treatment ( t4 , t6 , and t8 ) , compared to control explants ( c4 , c6 , and c8 ) . was performed to normalize the expression of nox4 protein to gapdh expression . upon tgf-2 treatment , nox4 expression was significantly upregulated at 6 hours ( t6 ) , and these levels significantly increased by 8 hours ( t8 ) . one - way anova , tukey 's post hoc test ( 2-tailed t - test ) * p < 0.05 and * * p < 0.01 . ( b ) a representative rt - pcr of nox14 mrna transcripts after 8 hours , in the absence ( c8 ) and presence ( t8 ) of tgf-2 . for each lane , the pcr products shown correspond to the expected base pair length ( nox1 , 201 base pairs [ bp ] ; nox2 , 149 bp ; nox3 , 234 bp ; nox4 , 191 bp , and gapdh , 287 bp ) . given that the expression level of nox4 was more pronounced at 8 hours after tgf-2 treatment , this time - point was used to examine the spatial localization of nox4 in rat lens epithelial cells using immunofluorescence and confocal microscopy ( fig . some punctate labeling was observable , and appeared to localize throughout the cytosol ( fig . this was in contrast to the two distinct expression patterns of nox4 seen at 8 hours following tgf-2 treatment ( fig . 2b ) . specifically , increased nox4 expression was seen throughout all tgf-2treated epithelial cells at 8 hours , with more pronounced punctate labeling in the cell nuclei and perinuclear areas . interestingly , nox4 localization in lens epithelial cells treated for longer periods with tgf-2 , at 48 hours , was predominantly cytosolic ( fig . 2e ) , which was first detectable by 24 hours after tgf-2 treatment ( data not shown ) . at 48 hours treatment , nox4 appeared to colocalize with particular areas of the -sma reactive stress fibers ( see fig . this colocalization of nox4 and -sma has been observed in other models of tgf-driven emt . interestingly , not all cells with a strong nox4 label colabeled with -sma ( see fig . similar to untreated cells at 8 hours , untreated cells at 48 hours yielded negligible nox4 expression and -sma  positive stress fiber formation . transforming growth factor-2 induces variable nox4 localization patterns in lens epithelial cells with the progression of tgf- induced emt . nox4 ( red ) was merged with the cell nuclear hoechst dye ( blue ) . untreated explants at 8 ( a ) and 48 ( c ) hours exhibited little nox4 expression . explants treated with tgf-2 ( + tgf-2 ) exhibited a stronger distinct punctate label by 8 hours ( b ) that primarily localized to the perinuclear regions of the cells . at 48 hours ( d ) , this label became more pronounced throughout the cytosol . by 48 hours compared to control explants ( e ) , epithelial cells treated with tgf-2 also labeled for -sma ( green ; [ f ] , with -sma labeled - stress fibres colocalizing with nox4 ( shown at higher magnification , inset 1 ) . punctate cytosolic distribution of nox4 , independent of stress fibers , also was apparent in some cells ( [ f ] , shown at higher magnification , inset 2 ) . scale bar : 40 and 20 m for the inserts . to validate the specificity of the nox4 label seen in response to tgf-2 treatment , we repeated our immunolabeling experiments with the pan - nadph oxidase inhibitor vas2870 ( fig . 3 ) . inhibition of nadph oxidase blocked nox4 protein expression in lens epithelial cells in response to tgf-2 treatment ( fig . 3i , inset ) when compared to tgf-2treated cells without the inhibitor ( see fig . we next determined whether this tgf-2dependent expression of nox4 was accompanied with nadph oxidase activity , using dhe fluorescence microscopy to assay for ros production in lens epithelial cells . as nox4 was first detected between 6 and 8 hours after tgf-2 treatment , dhe reactivity was assayed before and at these time points . reassuringly , nox4 expression was accompanied by a significant increase in nadph oxidase specific activity ( dhe oxidation ) in tgf-2treated lens explants ( figs . 3 g , 3h ) compared to the control explants not treated with tgf-2 ( figs . the ros probe , dhe , was used to assess increases in ros following tgf-2 treatment . in untreated explants , basal levels of ros were observable ( a  d ) . importantly , tgf-2 treated explants yielded noticeable increases in ros by 6 hours , that increased up to 8 hours ( h ) . treatment with vas2870 blocked tgf-2induced ros ( i ) as shown by diminished fluorescence intensity , with less ros produced . this was in conjunction to vas2870 also blocking nox4 protein expression ( see [ i ] inset ) . representative images from 3 independent experiments , consisting of at least 2 replicates for each treatment group . scale bar : 100 m ( a  i ) and 20m for ( inset [ i ] ) . total fluorescence intensity also was calculated using imagej ( j ) , with significant increases after 6 and 8 hours , effectively blocked by vas2870 . one - way anova , tukey 's post hoc test ( 2-tailed t - test ) * * further earlier time points that did not exhibit nox4 expression ( 2 and 4 hours ) did not produce a dhe label , either in the presence or absence of tgf-2 ( fig . 3 ) . in addition , when tgf-driven nox4 expression was inhibited with vas2870 ( see also fig . 3i ) . given that we have shown nox4 expression and activity is induced in lens epithelial cells and that we could inhibit nox4 expression and concomitant activity with vas2870 , we next investigated the role nox4 had in the development of tgf-dependent lens emt . the development of tgf-2induced lens emt can be followed morphologically in lens explants using phase - contrast microscopy . in our experimental model , 4b ) , and from day 3 exhibit progressive cell loss indicated by acellular patches ( fig . 4c , asterisk ) , cellular blebbing ( fig . 4d ) . interestingly , in explants treated with tgf-2 in the presence of vas2870 , cellular loss was significantly delayed by day 3 and most pronounced at 5 days after tgf-2 treatment ( fig . 4i ; day 3 , + tgf vs. + tgf/vas , p = 0.0001 and day 5 + tgf vs. + tgf/vas , p = 0.005 ) . moreover , cells in these explants did not appear to undergo tgf-2dependent cellular elongation , with cells remaining on day 5 of culture , exhibiting a typical uniformly packed lens epithelial phenotype ( fig . vas2870-alone treatment had no effect on e - cadherin labeling ( 5a ) . moreover , consistent with the absence of cell elongation earlier in the culture period , indicating the absence of myofibroblastic cells that are thought to promote lens capsular wrinkling , under these conditions there was no apparent capsular wrinkling ( fig . phase contrast images of explants at days 2 ( b , f ) , 3 ( c , g ) , and 5 ( a , e , d , h ) . explants treated with tgf-2 alone ( b  d ) underwent morphologic changes associated with emt compared to untreated cells ( a ) . explants treated with vas2870 and tgf-2 ( f  h ) showed a delayed emt response , with less cell elongation apparent by day 2 ( f ) , reduced cell loss by day 3 ( g ) and the retention of epithelial - like cells by day 5 ( h ) . note that , vas2870 alone ( e ) had little effect on cells treated up to 5 days of culture . ( i ) percentage cell loss was calculated using the threshold feature of imagej in relation to areas of bare lens capsule , with significant cell loss in tgf-2treated explants from day 3 , which was abrogated by application of vas2870 . two - way anova , tukey 's post hoc test ( 2-tailed t - test ) * * p < 0.01 and * * * * p < 0.0001 . explants were treated with vas2870 ( a ) , or cotreated with tgf-2 and vas2870 ( b ) and fixed at 5 days of culture . immunolabeling for the membrane marker e - cadherin ( green ) was performed and nuclei were counterstained with hoechst ( blue ) . in cells treated only with vas2870 ( a ) , e - cadherin localized to the cell membrane and there was no apparent cell loss . cotreatment with tgf-2 and vas2870 promoted increased cell survival and the maintenance of a membranous e - cadherin label ( b ) . explants cultured for 24 hours in the presence of tgf-2 ( a  c ) , or tgf-2 and vas2870 ( d  f ) were immunolabeled for nox4 ( [ b , e ] , red ) , -sma ( green ; [ c , f ] ) and counterstained with hoechst dye ( a , d ) . compared to explants treated with tgf alone ( a  c ) , vas2870 was shown to block tgf-2-induced nox4 ( e ) and -sma ( f ) expression . ( g ) protein lysates also were collected from explants treated with and without tgf-2 and vas2870 and western blotting demonstrated similar reductions in nox4 and -sma expression in the presence of vas2870 at 24 and 48 hours . 2d ) to localize with some -sma  reactive stress fibers , it was proposed that nox4 may have a role in -sma accumulation . cells treated with both vas2870 and tgf-2 yielded undetectable nox4 expression ( figs . 6b , 6 g ) , compared to explants treated with tgf-2 alone ( figs . 6e , 6 g ) , and in the process halted the progression of emt , with little to no detectable labeling for -sma expression in cells ( fig . 6 g ) that retained their epithelial - like appearance ( see hoechst staining , figs . nox4 expression in tgf-driven lens emt was assayed using immunolabeling of lens epithelial explant lysates . specifically , we determined nox4 expression using western blotting at different time points ( 0 , 2 , 4 , 6 , and 8 hours ) . 1 ) , and this level was significantly different to that of the untreated control at the same time period ( fig . expression of nox4 also was significantly more pronounced at 8 hours to that observed at 6 hours ( fig . 8 hours after tgf-2 treatment , the level of nox4 protein expression was approximately 2.5-fold higher to that of the matching control without tgf-2 ( fig . this increase in nox4 protein expression also was accompanied by an upregulation in nox4 mrna transcription in tgf-2treated explants compared to untreated explants at 8 hours ( fig . transforming growth factor-2 induces an increase in nox4 expression in lens epithelial explants : western blot . ( a ) representative western blot depicting the time - course ( 4 , 6 , and 8 hours ) of nox4 expression following tgf-2 treatment ( t4 , t6 , and t8 ) , compared to control explants ( c4 , c6 , and c8 ) . was performed to normalize the expression of nox4 protein to gapdh expression . upon tgf-2 treatment , nox4 expression was significantly upregulated at 6 hours ( t6 ) , and these levels significantly increased by 8 hours ( t8 ) . one - way anova , tukey 's post hoc test ( 2-tailed t - test ) * p < 0.05 and * * p < 0.01 . ( b ) a representative rt - pcr of nox14 mrna transcripts after 8 hours , in the absence ( c8 ) and presence ( t8 ) of tgf-2 . for each lane , the pcr products shown correspond to the expected base pair length ( nox1 , 201 base pairs [ bp ] ; nox2 , 149 bp ; nox3 , 234 bp ; nox4 , 191 bp , and gapdh , 287 bp ) . given that the expression level of nox4 was more pronounced at 8 hours after tgf-2 treatment , this time - point was used to examine the spatial localization of nox4 in rat lens epithelial cells using immunofluorescence and confocal microscopy ( fig . some punctate labeling was observable , and appeared to localize throughout the cytosol ( fig . this was in contrast to the two distinct expression patterns of nox4 seen at 8 hours following tgf-2 treatment ( fig . specifically , increased nox4 expression was seen throughout all tgf-2treated epithelial cells at 8 hours , with more pronounced punctate labeling in the cell nuclei and perinuclear areas . interestingly , nox4 localization in lens epithelial cells treated for longer periods with tgf-2 , at 48 hours , was predominantly cytosolic ( fig . 2e ) , which was first detectable by 24 hours after tgf-2 treatment ( data not shown ) . at 48 hours treatment , nox4 appeared to colocalize with particular areas of the -sma reactive stress fibers ( see fig . this colocalization of nox4 and -sma has been observed in other models of tgf-driven emt . interestingly , not all cells with a strong nox4 label colabeled with -sma ( see fig . 2 , inset 2 ) . similar to untreated cells at 8 hours , untreated cells at 48 hours yielded negligible nox4 expression and -sma  positive stress fiber formation . transforming growth factor-2 induces variable nox4 localization patterns in lens epithelial cells with the progression of tgf- induced emt . nox4 ( red ) was merged with the cell nuclear hoechst dye ( blue ) . untreated explants at 8 ( a ) and 48 ( c ) hours exhibited little nox4 expression . explants treated with tgf-2 ( + tgf-2 ) exhibited a stronger distinct punctate label by 8 hours ( b ) that primarily localized to the perinuclear regions of the cells . at 48 hours ( d ) , this label became more pronounced throughout the cytosol . by 48 hours compared to control explants ( e ) , epithelial cells treated with tgf-2 also labeled for -sma ( green ; [ f ] , with -sma labeled - stress fibres colocalizing with nox4 ( shown at higher magnification , inset 1 ) . punctate cytosolic distribution of nox4 , independent of stress fibers , also was apparent in some cells ( [ f ] , shown at higher magnification , inset 2 ) . to validate the specificity of the nox4 label seen in response to tgf-2 treatment , we repeated our immunolabeling experiments with the pan - nadph oxidase inhibitor vas2870 ( fig . 3 ) . inhibition of nadph oxidase blocked nox4 protein expression in lens epithelial cells in response to tgf-2 treatment ( fig . 3i , inset ) when compared to tgf-2treated cells without the inhibitor ( see fig . we next determined whether this tgf-2dependent expression of nox4 was accompanied with nadph oxidase activity , using dhe fluorescence microscopy to assay for ros production in lens epithelial cells . as nox4 was first detected between 6 and 8 hours after tgf-2 treatment , dhe reactivity was assayed before and at these time points . reassuringly , nox4 expression was accompanied by a significant increase in nadph oxidase specific activity ( dhe oxidation ) in tgf-2treated lens explants ( figs . 3 g , 3h ) compared to the control explants not treated with tgf-2 ( figs . the ros probe , dhe , was used to assess increases in ros following tgf-2 treatment . in untreated explants , basal levels of ros were observable ( a  d ) . importantly , tgf-2 treated explants yielded noticeable increases in ros by 6 hours , that increased up to 8 hours ( h ) . treatment with vas2870 blocked tgf-2induced ros ( i ) as shown by diminished fluorescence intensity , with less ros produced . this was in conjunction to vas2870 also blocking nox4 protein expression ( see [ i ] inset ) . representative images from 3 independent experiments , consisting of at least 2 replicates for each treatment group . scale bar : 100 m ( a  i ) and 20m for ( inset [ i ] ) . total fluorescence intensity also was calculated using imagej ( j ) , with significant increases after 6 and 8 hours , effectively blocked by vas2870 . one - way anova , tukey 's post hoc test ( 2-tailed t - test ) * * * p < 0.001 and * * * * p < 0.0001 . further earlier time points that did not exhibit nox4 expression ( 2 and 4 hours ) did not produce a dhe label , either in the presence or absence of tgf-2 ( fig . 3 ) . in addition , when tgf-driven nox4 expression was inhibited with vas2870 ( see also fig . given that we have shown nox4 expression and activity is induced in lens epithelial cells and that we could inhibit nox4 expression and concomitant activity with vas2870 , we next investigated the role nox4 had in the development of tgf-dependent lens emt . the development of tgf-2induced lens emt can be followed morphologically in lens explants using phase - contrast microscopy . in our experimental model , 4b ) , and from day 3 exhibit progressive cell loss indicated by acellular patches ( fig . 4d ) . interestingly , in explants treated with tgf-2 in the presence of vas2870 , cellular loss was significantly delayed by day 3 and most pronounced at 5 days after tgf-2 treatment ( fig . 4i ; day 3 , + tgf vs. + tgf/vas , p = 0.0001 and day 5 + tgf vs. + tgf/vas , p = 0.005 ) . moreover , cells in these explants did not appear to undergo tgf-2dependent cellular elongation , with cells remaining on day 5 of culture , exhibiting a typical uniformly packed lens epithelial phenotype ( fig . moreover , consistent with the absence of cell elongation earlier in the culture period , indicating the absence of myofibroblastic cells that are thought to promote lens capsular wrinkling , under these conditions there was no apparent capsular wrinkling ( fig . phase contrast images of explants at days 2 ( b , f ) , 3 ( c , g ) , and 5 ( a , e , d , h ) . explants treated with tgf-2 alone ( b  d ) underwent morphologic changes associated with emt compared to untreated cells ( a ) . explants treated with vas2870 and tgf-2 ( f  h ) showed a delayed emt response , with less cell elongation apparent by day 2 ( f ) , reduced cell loss by day 3 ( g ) and the retention of epithelial - like cells by day 5 ( h ) . note that , vas2870 alone ( e ) had little effect on cells treated up to 5 days of culture . ( i ) percentage cell loss was calculated using the threshold feature of imagej in relation to areas of bare lens capsule , with significant cell loss in tgf-2treated explants from day 3 , which was abrogated by application of vas2870 . two - way anova , tukey 's post hoc test ( 2-tailed t - test ) * * p < 0.01 and * * * * p < 0.0001 . explants were treated with vas2870 ( a ) , or cotreated with tgf-2 and vas2870 ( b ) and fixed at 5 days of culture . immunolabeling for the membrane marker e - cadherin ( green ) was performed and nuclei were counterstained with hoechst ( blue ) . in cells treated only with vas2870 ( a ) , e - cadherin localized to the cell membrane and there was no apparent cell loss . cotreatment with tgf-2 and vas2870 promoted increased cell survival and the maintenance of a membranous e - cadherin label ( b ) . explants cultured for 24 hours in the presence of tgf-2 ( a  c ) , or tgf-2 and vas2870 ( d  f ) were immunolabeled for nox4 ( [ b , e ] , red ) , -sma ( green ; [ c , f ] ) and counterstained with hoechst dye ( a , d ) . compared to explants treated with tgf alone ( a  c ) , vas2870 was shown to block tgf-2-induced nox4 ( e ) and -sma ( f ) expression . ( g ) protein lysates also were collected from explants treated with and without tgf-2 and vas2870 and western blotting demonstrated similar reductions in nox4 and -sma expression in the presence of vas2870 at 24 and 48 hours . 2d ) to localize with some -sma  reactive stress fibers , it was proposed that nox4 may have a role in -sma accumulation . cells treated with both vas2870 and tgf-2 yielded undetectable nox4 expression ( figs . 6b , 6 g ) , compared to explants treated with tgf-2 alone ( figs . 6e , 6 g ) , and in the process halted the progression of emt , with little to no detectable labeling for -sma expression in cells ( fig . 6 g ) that retained their epithelial - like appearance ( see hoechst staining , figs . in the present study , for the first time , we describe the novel temporal and spatial expression of nox4 and characterize its activity in the rat lens explant system following tgf- treatment . we demonstrated that nox4 is the only nox homologue expressed in rat lens epithelial explants , and is responsive to tgf , consistent with other studies . nox4 inhibition using a pan - nadph oxidase inhibitor slowed the progression of emt through the promotion of cell survival . furthermore , we also impeded the expression of the known myofibroblastic emt marker , -sma , and subsequent development of capsular wrinkling . the remaining / surviving cells on the lens capsule following the nox4 blockade retain an epithelial - like phenotype and do not progress down the emt pathway , highlighting an important role for nox4 in tgf-dependent emt in the lens . reactive oxygen species are known to have a key role in the development of cataract . these antioxidants include the glutathione synthesis machinery , the detoxification enzyme peroxiredoxin , and glutaredoxins , that are responsible for the maintenance of the thiol redox status of lens proteins , including the crystallins . previous studies have shown the indirect role of ros in the development of tgf-dependent cataract in rats . in these studies , addition of the antioxidant gsh was shown to strongly suppress tgf-induced opacification and subcapsular plaque formation in whole rat lens cultures . furthermore , microarray studies using tgf-treated human lens cell lines highlighted that nox4 was not only induced in response to tgf- , but was one of the highest induced transcripts in these cultured human lens epithelial cells compared to controls . while we demonstrate nox4 to be involved in tgf-dependent emt in lens , it has previously been reported to be involved in a number of other different models of emt . for example , in breast cancer cells , tgf- has been shown to induce nox4 expression and activity ( by measuring the production of ros in the presence of a nadph oxidase inhibitor ) . furthermore , in this earlier study , tgf-dependent nox4 expression and activity was dependent on the smad signaling pathway , given inhibition of this pathway ( via sirna or pharmacological inhibition ) inhibited nox4 labeling / activity and abolished the development of an emt . additionally , blocking nadph oxidase activity alone ( either pharmacologically or by sirna ) , also prevented emt in breast cancer cells , highlighting that nox4 expression and activity was responsible for the development of emt in breast cancer cells . it is likely that tgf- signals through the smad pathway to induce nox4 expression and activity in lens . in future studies , it would be of great interest to determine whether direct or indirect tgf-smad signaling contributes to the differential nuclear ( at 8 hours ) and cytosolic ( 48 hours ) nox4 labeling we observed here . furthermore , nox4 expressed at later time points in the emt process may directly influence the actin cytoskeleton with which it is colocalized . novel studies in the kidney have highlighted the role of nox4 in actin cytoskeleton rearrangement , leading to regulation of focal adhesion turnover and vsmc migration . members of the rho gtpase family are strongly associated with the formation of the actin cytoskeleton . ascertained that rhoa / rock activation is upstream of poldip2 and nox4 in tgf- signaling in the kidney , and controls myofibroblast activation . indeed , in the rat lens explant cellular blebbing is an indirect indication of apoptosis , where cells are physically dismantled from within by modulation of the actin cytoskeleton . this process is mediated by members of the proapoptotic caspase family , where the plasma membrane is weakened allowing the cytosol to protrude from the cell . caspases have been shown to cleave and activate rho kinase ( rock i ) , a downstream effector of rhoa signaling . activation of rock i leads to the formation of actin stress fibers , cell contraction and dynamic membrane blebbing induced by active rock i. therefore , it is feasible that the changes seen in the tgf-driven emt in the lens may signal at least in part through the rhoa / rock pathway via nox4 . reflective of its multiple roles in proinflammatory responses , migration , differentiation and apoptosis , nox4-derived ros appear enigmatic in their downstream targets . using tgf-stimulated human aortic muscle cells , martin - garrido et al . found that silencing of nox4 abolished the formation of stress fibers and -sma accumulation in a mapk / p38-dependent manner . this is in contrast to research using fibroblastic cells that report the c - jun n - terminal kinase ( jnk pathway ) as capable of regulating -sma accumulation in a nox4-dependent fashion . together , these findings from other fibrotic models and the lens suggest that the progressive accumulation of -sma is the result of a heterogeneous process that converges on the -sma promoter , and that nox4 may act as a common mediator of these pathways . more broadly , this is indirect evidence to the notion that redox signaling is capable of serving as a nexus between parallel signaling pathways , such as smads and mapks . the present study shows for the first time to our knowledge that nox4 has a role in tgf-driven lens emt . in particular , nox4 appears to have a crucial role in the regulation of -sma , potentially apoptosis , cell elongation and capsular wrinkling . the findings of this study provide new avenues for the potential treatment of cataract and the prevention of secondary cataract , given tgf-driven emt is integral to the development of some cataracts in humans . future studies will concentrate on expanding these findings in genetic models of tgf-dependent cataract , ascertaining the upstream events crucial to nox4 expression and delineating the specific targets of ros during lens emt .