balb / c mice were immunized with purified human pcna protein overexpressed in bl21 cells .
after 3 injections of 0.1 mg protein / mouse mixed with poly i - poly c polymer , the spleen cells of the responding animals were fused with sp2/0-ag14 .
growing clones were screened by elisa using microtiter plates coated with pure pcna protein ( 0.5 g / well ) and by cell staining using fixed hela cells .
all antibodies were purified from either ascitic fluid or hybridoma supernatant on protein g sepharose ( ge healthcare ) .
they were subsequently dialysed against pbs and concentrated with centrifugal filtration units ( merck millipore ) to obtain antibody samples of 25 mg / ml .
purified antibody ( 1 mg ) in pbs was incubated with a 20-fold molar excess of sulfosuccinimidyl-4-(n - maleimidomethyl)- cyclohexane-1-carboxylate ( sulfo - smcc ; thermo scientific ) for 1 h at room temperature .
excess crosslinker was removed using a zeba desalt spin column ( 2 ml ; thermo scientific ) .
the antibodies recovered in the flow - trough were mixed with a 5- to 15-fold molar excess of the nls - encoding synthetic peptide ( cagpkkkrkved ) containing a cysteine residue at the n - terminal end .
after incubation for 1 h at room temperature , the antibody - nls peptide conjugates were purified on desalting columns as described above and stored at 4c at a concentration of 2 mg / ml .
the hela , caski , mel501 and u2os cell lines were grown according to atcc guidelines and maintained in dulbecco s modified eagle s tissue culture medium ( dmem ; life technologies ) supplemented with l - glutamine ( 2 mm ) , gentamicin ( 50 g / ml ) and 10% heat - inactivated fetal calf serum at 37c in a humidified 5% co2 atmosphere .
transduction experiments with purified mabs were performed according to the neon transfection system protocol ( life technologies ) .
typically , 10 cells in r buffer ( provided in the kit ) were mixed with 25 g of antibody in a total volume of 12 l .
the mixture was then transferred in a neon pipette tip chamber ( 10 l ) and subjected to electroporation in the system pipette station by applying the following optimized parameters : 1550 v , 10 ms and 3 pulses .
the treated samples were deposited into 24-well plates containing sterile glass coverslips in 500 l pre - warmed culture medium without the antibiotic .
after incubation for 24 h at 37c , the culture medium was replaced with complete medium . where indicated , the cells were irradiated with 20 j / m2 uvc 8 h before harvesting . for the electroporation of sirna ( final concentration 50 nmol / l ) , settings were 1005 v , 35 ms and 2 pulses .
the electroporated cells were fixed at the indicated time with 4% paraformaldehyde ( w / v ) for 30 min and washed twice with pbs .
after permeabilization with 0.2% triton x100 for 5 min , they were incubated for 1 h with alexafluor 488 or 568 labeled - anti - mouse immunoglobulins ( molecular probes , eugene , usa ) .
following several washes with pbs , the coverslips were mounted with 4,6-diamino-2 phenylindole ( dapi ) fluoromount - g ( southernbiotech ) .
the cells were imaged with a leica dm5500 microscope equipped with fitc and cy3 filters .
images were collected with a 63  or a 100  objective and analyzed with photoshop software . for the quantification of the delivered antibodies
, cells were electroporated in the presence of varying amounts ( 0.5 to 5 g ) of antibody and incubated for 24 h in 24-well plates as above .
following recovery by trypsinization and counting , the cells were lysed in ripa buffer and soluble extracts corresponding to 5.10 cells were analyzed by western blotting , in parallel with calibrated antibody preparations .
the heavy chain content of these extracts was finally quantitated after incubation with horseradish peroxidase - conjugated anti - mouse globulins and ecl reagent ( ge healthcare ) and analysis with the imagequantlas 4000 imager ( ge healthcare ) .
the analysis of the binding activity of the anti - pcna antibodies was essentially performed as above , except that extracts from untreated cells were used .
the actin polypeptide was revealed with rabbit polyclonal antibody a206 ( sigma - aldrich ) .
briefly , caski cells were plated in triplicate in 12-well plates ( 10 cells / well ) and , after 24 h , treated with sirna ( 25 pmol ) or antibody ( 3 g ) as described above . after 48 h of incubation at 37c ,
the cells were harvested by trypsinization and 1/30 of the pooled cell suspension was replated in triplicate in 60 mm - diameter tissue culture dishes .
after 67 d of incubation in complete medium , the growing cells were counted manually .
balb / c mice were immunized with purified human pcna protein overexpressed in bl21 cells .
after 3 injections of 0.1 mg protein / mouse mixed with poly i - poly c polymer , the spleen cells of the responding animals were fused with sp2/0-ag14 .
growing clones were screened by elisa using microtiter plates coated with pure pcna protein ( 0.5 g / well ) and by cell staining using fixed hela cells .
all antibodies were purified from either ascitic fluid or hybridoma supernatant on protein g sepharose ( ge healthcare ) .
they were subsequently dialysed against pbs and concentrated with centrifugal filtration units ( merck millipore ) to obtain antibody samples of 25 mg / ml .
purified antibody ( 1 mg ) in pbs was incubated with a 20-fold molar excess of sulfosuccinimidyl-4-(n - maleimidomethyl)- cyclohexane-1-carboxylate ( sulfo - smcc ; thermo scientific ) for 1 h at room temperature .
excess crosslinker was removed using a zeba desalt spin column ( 2 ml ; thermo scientific ) .
the antibodies recovered in the flow - trough were mixed with a 5- to 15-fold molar excess of the nls - encoding synthetic peptide ( cagpkkkrkved ) containing a cysteine residue at the n - terminal end .
after incubation for 1 h at room temperature , the antibody - nls peptide conjugates were purified on desalting columns as described above and stored at 4c at a concentration of 2 mg / ml .
the hela , caski , mel501 and u2os cell lines were grown according to atcc guidelines and maintained in dulbecco s modified eagle s tissue culture medium ( dmem ; life technologies ) supplemented with l - glutamine ( 2 mm ) , gentamicin ( 50 g / ml ) and 10% heat - inactivated fetal calf serum at 37c in a humidified 5% co2 atmosphere .
transduction experiments with purified mabs were performed according to the neon transfection system protocol ( life technologies ) .
typically , 10 cells in r buffer ( provided in the kit ) were mixed with 25 g of antibody in a total volume of 12 l .
the mixture was then transferred in a neon pipette tip chamber ( 10 l ) and subjected to electroporation in the system pipette station by applying the following optimized parameters : 1550 v , 10 ms and 3 pulses .
the treated samples were deposited into 24-well plates containing sterile glass coverslips in 500 l pre - warmed culture medium without the antibiotic .
after incubation for 24 h at 37c , the culture medium was replaced with complete medium . where indicated , the cells were irradiated with 20 j / m2 uvc 8 h before harvesting . for the electroporation of sirna ( final concentration 50 nmol / l ) , settings were 1005 v , 35 ms and 2 pulses .
the electroporated cells were fixed at the indicated time with 4% paraformaldehyde ( w / v ) for 30 min and washed twice with pbs .
after permeabilization with 0.2% triton x100 for 5 min , they were incubated for 1 h with alexafluor 488 or 568 labeled - anti - mouse immunoglobulins ( molecular probes , eugene , usa ) .
following several washes with pbs , the coverslips were mounted with 4,6-diamino-2 phenylindole ( dapi ) fluoromount - g ( southernbiotech ) .
the cells were imaged with a leica dm5500 microscope equipped with fitc and cy3 filters .
images were collected with a 63  or a 100  objective and analyzed with photoshop software .
for the quantification of the delivered antibodies , cells were electroporated in the presence of varying amounts ( 0.5 to 5 g ) of antibody and incubated for 24 h in 24-well plates as above .
following recovery by trypsinization and counting , the cells were lysed in ripa buffer and soluble extracts corresponding to 5.10 cells were analyzed by western blotting , in parallel with calibrated antibody preparations .
the heavy chain content of these extracts was finally quantitated after incubation with horseradish peroxidase - conjugated anti - mouse globulins and ecl reagent ( ge healthcare ) and analysis with the imagequantlas 4000 imager ( ge healthcare ) .
the analysis of the binding activity of the anti - pcna antibodies was essentially performed as above , except that extracts from untreated cells were used .
the actin polypeptide was revealed with rabbit polyclonal antibody a206 ( sigma - aldrich ) .
, caski cells were plated in triplicate in 12-well plates ( 10 cells / well ) and , after 24 h , treated with sirna ( 25 pmol ) or antibody ( 3 g ) as described above . after 48 h of incubation at 37c ,
the cells were harvested by trypsinization and 1/30 of the pooled cell suspension was replated in triplicate in 60 mm - diameter tissue culture dishes .
after 67 d of incubation in complete medium , the growing cells were counted manually .
 antibodies are valuable tools for functional studies in vitro , but their use in living cells remains challenging because they do not naturally cross the cell membrane . here , we present a simple and highly efficient method for the intracytoplasmic delivery of any antibody into cultured cells . by following the fate of monoclonal antibodies that bind to nuclear antigens , it was possible to image endogenous targets and to show that inhibitory antibodies are able to induce cell growth suppression or cell death . 
 our electrotransfer system allowed the cancer cells we studied to be transduced without loss of viability and may have applications for a variety of intracellular immuno - interventions . 
Materials and Methods
Generation and purification of anti-PCNA monoclonal antibodies
Preparation of antibody-NLS peptide conjugates
Cell culture and electroporation
Immunofluorescence microscopy
Western blot analysis
Clonogenic survival assay
Supplementary Material