mef ( wt , nemo , ikk , ikk , ikk , and parkin ) cells were cultured in dulbecco 's modified eagle 's medium ( dmem ; invitrogen , carlsbad , nm , usa ) , supplemented with 10% fetal calf serum , 2  mm l - glutamine , 50  iu penicillin , and 50  g / ml streptomycin in 5% co2 , at 37  c . mef cells were transfected using lipofectamine2000 ( invitrogen ) with plasmids encoding for either gfp or park2 plasmid . sts was purchased from sigma ( la chapelle - sur - erdre , france ) . cells were also cotransfected with gfp , and the percentage of dying cells was assessed using a propidium iodide nuclear probe ( pi , molecular probes , life technology , cergy pontoise , france ) . cells were analyzed by flow cytometry ( fc500 flow cytometer , beckman coulter , villepinte , france ) . total rna was isolated from the different mef cell lines after 24  h of transfection . cdna synthesis was performed using the affinityscript qpcr cdna synthesis kit ( stratagene , agilent technologies , paris , france ) with 1  g of total rna and random primers . the resulting complementary - dna product was expanded using the absolute blue qpcr sybr green rox mix ( thermo scientific , illkirch , france ) with specific primers for murine opa1 ( forward : 5-accacaggaagctcaagaaatc-3 , reverse : 5-tctgatctccaaccacaacaac-3 ) . sample input was normalized through quantification of the transcript levels of the housekeeping genes gapdh ( forward : 5- ggctcatgaccacagtcc-3 , reverse : 5-gcagggatgatgttctgg-3 ) and rps18 ( forward : 5-ggaagtacagccaggttctg-3 , reverse : 5-gtggtcttggtgtgctgac-3 ) . detection was performed with the abi prism 7900ht sequence detection system ( applied biosystems , saint aubin , france ) . threshold cycle ( ct ) values were obtained for each gene using the instrument software and auto - ct function . total lysates were extracted using 1% np40 ( 150  mm nacl , 50  mm tris hcl ph 7.4 , 10  mm edta ) supplemented by a cocktail of antiproteases and antiphosphatases . a total of 20  g of proteins from each point were resolved by sds - nupage ( 412% bis - tris gels , novex , les ulis , france ) and transferred to nitrocellulose membranes ( amersham biosciences , les ulis , france ) . nonspecific sites were blocked by incubation with 5% milk for 1  h at room temperature , and the membranes were then incubated with mouse monoclonal anti - opa1 ( bd biosciences pharmingen , le pont de claix , france ; clone 18 ) , anti - parkin ( millipore , le pont de claix , france ) , and rabbit anti - bax ( n-20 , from santa - cruz , le pont de claix , france ) . antibodies specific for ikk , ikk , and nemo were purchased from cell signaling ( st quentin fallavier , france ) . equal protein loading was assessed by probing the membranes with anti - actin ( millipore ) and anti - hsp60 monoclonal antibodies ( stressgen , st quentin fallavier , france ) . membranes were treated with horseradish peroxidase - linked goat anti - mouse or anti - rabbit secondary antibodies ( amersham biosciences ) . immunoreactive proteins were detected and quantified by enhanced chemiluminescence ( amersham biosciences ) using a ccd camera ( gbox , syngene , ozyme , france ) . mef cells were grown on coverslips and fixed with 4% paraformaldehyde ( sigma - aldrich , st quentin fallavier , france ) . cells were incubated for 1  h in blocking buffer ( 2% bovine serum albumin in phosphate - buffered saline ) and then overnight with a mouse monoclonal anti - cytochrome c ( bd biosciences , st quentin fallavier , france ; clone 6h2.b4 ) , or goat anti - hsp60 ( santa cruz ) antibody . cells were washed and incubated for 2  h with alexa fluor secondary anti - mouse , anti - rabbit , or anti - goat antibodies ( molecular probes ) . images were acquired with a zeiss lsm 510 confocal microscope equipped with an oil - immersion fluorescence objective ( carl zeiss , inc . , prigny , france ) . cells were also stained with 100  nm mitotracker green ( invitrogen ) for 30  min . images were then analyzed using the imagej software ( nih , bethesda , md , usa ) . deconvolution was performed by subtracting a gaussian blur (  : 12 ) to the original image . deconvolved images were then processed with a certain threshold to obtain a binary image of the mitochondrial network . the binary image was then analyzed using the analyze particle function of imagej to determine the number and the size of mitochondria in individual cells . mef ( wt , nemo , ikk , ikk , ikk , and parkin ) cells were cultured in dulbecco 's modified eagle 's medium ( dmem ; invitrogen , carlsbad , nm , usa ) , supplemented with 10% fetal calf serum , 2  mm l - glutamine , 50  iu penicillin , and 50  g / ml streptomycin in 5% co2 , at 37  c . mef cells were transfected using lipofectamine2000 ( invitrogen ) with plasmids encoding for either gfp or park2 plasmid . sts was purchased from sigma ( la chapelle - sur - erdre , france ) . cells were also cotransfected with gfp , and the percentage of dying cells was assessed using a propidium iodide nuclear probe ( pi , molecular probes , life technology , cergy pontoise , france ) . cells were analyzed by flow cytometry ( fc500 flow cytometer , beckman coulter , villepinte , france ) . total rna was isolated from the different mef cell lines after 24  h of transfection . cdna synthesis was performed using the affinityscript qpcr cdna synthesis kit ( stratagene , agilent technologies , paris , france ) with 1  g of total rna and random primers . the resulting complementary - dna product was expanded using the absolute blue qpcr sybr green rox mix ( thermo scientific , illkirch , france ) with specific primers for murine opa1 ( forward : 5-accacaggaagctcaagaaatc-3 , reverse : 5-tctgatctccaaccacaacaac-3 ) . sample input was normalized through quantification of the transcript levels of the housekeeping genes gapdh ( forward : 5- ggctcatgaccacagtcc-3 , reverse : 5-gcagggatgatgttctgg-3 ) and rps18 ( forward : 5-ggaagtacagccaggttctg-3 , reverse : 5-gtggtcttggtgtgctgac-3 ) . detection was performed with the abi prism 7900ht sequence detection system ( applied biosystems , saint aubin , france ) . threshold cycle ( ct ) values were obtained for each gene using the instrument software and auto - ct function . the relative levels of gene expression were determined using the delta  delta ct method . total lysates were extracted using 1% np40 ( 150  mm nacl , 50  mm tris hcl ph 7.4 , 10  mm edta ) supplemented by a cocktail of antiproteases and antiphosphatases . a total of 20  g of proteins from each point were resolved by sds - nupage ( 412% bis - tris gels , novex , les ulis , france ) and transferred to nitrocellulose membranes ( amersham biosciences , les ulis , france ) . nonspecific sites were blocked by incubation with 5% milk for 1  h at room temperature , and the membranes were then incubated with mouse monoclonal anti - opa1 ( bd biosciences pharmingen , le pont de claix , france ; clone 18 ) , anti - parkin ( millipore , le pont de claix , france ) , and rabbit anti - bax ( n-20 , from santa - cruz , le pont de claix , france ) . antibodies specific for ikk , ikk , and nemo were purchased from cell signaling ( st quentin fallavier , france ) . equal protein loading was assessed by probing the membranes with anti - actin ( millipore ) and anti - hsp60 monoclonal antibodies ( stressgen , st quentin fallavier , france ) . membranes were treated with horseradish peroxidase - linked goat anti - mouse or anti - rabbit secondary antibodies ( amersham biosciences ) . immunoreactive proteins were detected and quantified by enhanced chemiluminescence ( amersham biosciences ) using a ccd camera ( gbox , syngene , ozyme , france ) . mef cells were grown on coverslips and fixed with 4% paraformaldehyde ( sigma - aldrich , st quentin fallavier , france ) . cells were incubated for 1  h in blocking buffer ( 2% bovine serum albumin in phosphate - buffered saline ) and then overnight with a mouse monoclonal anti - cytochrome c ( bd biosciences , st quentin fallavier , france ; clone 6h2.b4 ) , or goat anti - hsp60 ( santa cruz ) antibody . cells were washed and incubated for 2  h with alexa fluor secondary anti - mouse , anti - rabbit , or anti - goat antibodies ( molecular probes ) . images were acquired with a zeiss lsm 510 confocal microscope equipped with an oil - immersion fluorescence objective ( carl zeiss , inc . , cells were also stained with 100  nm mitotracker green ( invitrogen ) for 30  min . images were then analyzed using the imagej software ( nih , bethesda , md , usa ) . deconvolution was performed by subtracting a gaussian blur (  : 12 ) to the original image . deconvolved images were then processed with a certain threshold to obtain a binary image of the mitochondrial network . the binary image was then analyzed using the analyze particle function of imagej to determine the number and the size of mitochondria in individual cells .
the optic atrophy 1 protein ( opa1 ) is a key element in the dynamics and morphology of mitochondria . 
 we demonstrated that the absence of ib kinase- , which is a key element of the nonclassical nf-b pathway , has an impact on the mitochondrial network morphology and opa1 expression . 
 in contrast , the absence of nf-b essential modulator ( nemo ) or ib kinase- , both of which are essential for the canonical nf-b pathway , has no impact on mitochondrial dynamics . whereas parkin has been reported to positively regulate the expression of opa1 through nemo , herein we found that park2 overexpression did not modify the expression of opa1 . 
 park2 expression reduced the levels of bax , and it prevented stress - induced cell death only in bak - deficient mouse embryonic fibroblast cells . 
 collectively , our results point out a role of the nonclassical nf-b pathway in the regulation of mitochondrial dynamics and opa1 expression .