From the Unité Mixte de Recherches 5124 CNRS,
Université Montpellier 2, Montpellier, France, ¶ Section on
Membrane Biology, Laboratory of Cellular and Molecular Biophysics,
NICHD, National Institutes of Health, Bethesda, Maryland 20892, and
** Medical Research Council, Laboratory of Molecular
Biology, Hills Road, Cambridge CB2 2QH, United Kingdom
Received for publication, September 17, 2002, and in revised form, October 22, 2002
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ABSTRACT |
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Cellular uptake of a family of cationic
cell-penetrating peptides (examples include Tat peptides and
penetratin) have been ascribed in the literature to a mechanism that
does not involve endocytosis. In this work we reevaluate the mechanisms
of cellular uptake of Tat 48-60 and (Arg)9. We
demonstrate here that cell fixation, even in mild conditions, leads to
the artifactual uptake of these peptides. Moreover, we show that flow
cytometry analysis cannot be used validly to evaluate cellular uptake
unless a step of trypsin digestion of the cell membrane-adsorbed
peptide is included in the protocol. Fluorescence microscopy on live
unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are
similar to the kinetics of endocytosis. Peptide uptake is inhibited by
incubation at low temperature and cellular ATP pool depletion. Similar
data were obtained for Tat-conjugated peptide nucleic acids. These data
are consistent with the involvement of endocytosis in the cellular
internalization of cell-penetrating peptides and their conjugates to
peptide nucleic acids.
During the last decade, several proteins, such as HIV-1 Tat,
Drosophila Antennapedia homeoprotein, and HSV-1 VP22 have
been shown to traverse the cell membrane by a process called protein transduction and to reach the nucleus while retaining their biological activity (1-5). Short "protein-transduction domains" are
responsible for the cellular uptake of these proteins (6, 7). Although the biological relevance of protein transduction remains to be understood, it has attracted much interest. Indeed, it was discovered that short peptides derived from protein-transduction domains (cell-penetrating peptides or
CPPs)1 can be internalized in
most cell types and, more importantly, allow the cellular delivery of
conjugated (or fused) biomolecules (8, 9). A wide range of biomolecules
such as antigenic peptides (10), peptide nucleic acids (11), antisense
oligonucleotides (12), full-length proteins (13-15), or even
nanoparticles (16) and liposomes (17) have been delivered this way.
Most peptide- and nucleic acid-based drugs are poorly taken up in
cells, and this is considered a major limitation in their development
as therapeutic agents (reviewed in refs. 18 and 19). Conjugation of
therapeutic agents to CPPs could thus become a strategy of choice to
improve their pharmacological properties.
The mechanism of internalization of CPPs and their cargo is not well
understood and has recently been the subject of controversies. It has
been described in the literature that internalization of these peptides
is not significantly inhibited by incubation at low temperature, by
depletion of the cellular ATP pool, or by inhibitors of endocytosis (7,
20-22). Moreover, structure-activity studies indicate that the
internalization of CPPs do not depend on its specific primary sequence,
which implies independence of receptor recognition (20, 23, 24). Based
on these results, it has been commonly accepted that the
internalization of CPPs do not involve endocytosis or specific protein
transporters. Instead, a direct transport through the lipid bilayer of
membranes has been proposed as a possible mechanism of translocation
(7, 20). If correct, this mechanism would require a radical revision of
current ideas on the properties of lipid bilayers, taking into account
the hydrophilic nature of CPPs such as Tat or (Arg)9 and the fact that there is no indication of increased membrane permeability in the presence of these peptides.
Most studies on the mechanism of CPP translocation
essentially rely on two techniques, namely fluorescence microscopy on
fixed cells and fluorescence-activated cell sorter (FACS) analysis. Recently it was shown that the fixation of cells with methanol induces
the artificial nuclear association of VP22 and histone H1 (25). This
raised concerns about the validity of histochemical detection of CPP
distribution in fixed cells.
We demonstrate here that cell fixation, even under mild conditions,
leads to the artifactual redistribution of CPP into the nucleus.
Moreover, these peptides bind strongly to the cell plasma membrane and
remain associated with cells even after repeated washings. As a
consequence, FACS analysis cannot be validly used to evaluate cellular
uptake unless a protease digestion step of the adsorbed CPP is included
in the protocol.
These findings have prompted us to reevaluate the mechanism of cellular
uptake of two widely used CPP, namely Tat 48-60 and (Arg)9, using as tools fluorescence microscopy on unfixed
cells and FACS analysis on trypsin-treated cells. Both sets of data are
consistent with the involvement of endocytosis in the cellular internalization of CPP and their conjugates to PNAs (peptide nucleic acids).
Peptide Synthesis and Labeling--
Synthesis of Tat peptide
with sequence Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro and
of (Arg)9 were carried out by solid phase on a Pioneer
peptide synthesizer (Applied Biosystems) following the Fmoc chemistry
protocol. A cysteine was added to the C-terminal end of the peptides to
provide a sulfhydryl group for ligation to a fluorochrome. Peptides
were purified by preparative HPLC and characterized by analytical HPLC
and matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) analysis (data not shown). Labeling with the fluorochrome
was performed by conjugation with a 10 molar excess of the
fluorochrome-maleimide derivatives (Molecular Probes) in 50 mM Tris-HCl buffer pH 7.2 for 4 h in the dark. Labeled
peptides were purified by semi-preparative HPLC, freeze-dried, and
resuspended in deionized water. Peptides were stored frozen at
Synthesis of Fluorescein-Tat-PNA and Fluorescein
PNA-Tat--
PNA-peptide conjugates were synthesized sequentially on
an ABI Pioneer peptide synthesizer (peptide part) and an ABI 380B DNA
synthesizer (PNA part) using a Fmoc-PAL-polyethylene glycol polystyrene support. For the Peptide-PNA construct, the peptide and PNA
parts were synthesized using the ABI 380B Synthesizer. Fmoc-PNA(Bhoc) monomers were obtained from Applied Biosystems, and Fmoc amino acids from Novabiochem with Pbf protection for Arg,
Trityl for Gln, and t-butyloxycarbonyl (Boc) for Lys.
Assembly of the Tat peptide sequence (GRKKRRQRRRP) was by the standard Fmoc procedure using
PyBOP/DIPEA/N,N-dimethylformamide coupling reactions (26). The anti-TAR PNA sequence (CTCCCAGGCTCA) (27) was
assembled also by the Fmoc procedure with PyAOP in
N,N-dimethylformamide as coupling agent and
DIPEA/2,6-lutidine (2:3) in N-methylpyrollidone as the base
(28). Peptide and PNA parts were joined by an AEEA linker
(2-aminoethoxy-2-ethoxyacetic acid, Applied Biosystems) using a
standard PNA coupling cycle. After assembly of the PNA-peptide or
peptide-PNA, one further coupling reaction was carried out with
6-carboxyfluorescein diacetate (Sigma, 10 eq) pre-activated with HATU
in the presence of 1 equivalent of DIPEA. After 10 min, a second
equivalent of DIPEA was added, and the mixture was added to the solid
support. After coupling, the acetyl esters were removed by treatment
with 20% piperidine/N,N-dimethylformamide, and
the conjugate was cleaved from the support in trifluoroacetic
acid /phenol/water/triisopropylsilane (85:10:2.5:2.5) for
4 h and precipitated with diethyl ether. After lyophilization from
a 0.1% trifluoroacetic acid solution in water, the conjugates were
purified by reversed-phase HPLC on a C18 column with
acetonitrile gradient in 0.1% trifluoroacetic acid in water. Product
masses determined by MALDI-TOF mass spectrometry agreed with expected
values (data not shown).
Cells and Cell Cultures--
HeLa cells were cultured as
exponentially growing subconfluent monolayers on 90-mm plates in RPMI
1640 medium (Invitrogen) supplemented with 10% (v/v) fetal calf
serum and 2 mM glutamine. CHO-K1 cells were cultured as
exponentially growing subconfluent monolayers on 25-cm2
culture flasks in F-12K medium (Invitrogen) supplemented with 10%
(v/v) fetal calf serum and 2 mM glutamine. Jurkat lymphoid cells were cultured in suspension in RPMI 1640 medium (Invitrogen) supplemented with 10% (v/v) fetal calf serum and 2 mM glutamine.
Fluorescence Microscopy--
Exponentially growing cells were
dissociated with a nonenzymatic cell dissociation medium (Sigma).
2.5 × 105 cells were plated and cultured overnight on
30-mm plates on a glass coverslip. The culture medium was discarded,
and the cells were washed with NaCl/Pi (pH 7.3).
NaCl/Pi was discarded, and the cell monolayer was incubated
with the peptides dissolved in Opti-MEM at the appropriate
concentration. Subsequently, cells were rinsed three times for 5 min
with NaCl/Pi for the observation of the living cells. For
the fixed cells, the protocol was the same, but, in addition, they were
fixed in 3.7% (v/v) formaldehyde in NaCl/Pi for 5 min at
room temperature. For direct detection of fluorescein-labeled peptides,
cells were washed three times after fixation in NaCl/Pi at
room temperature and washed again with NaCl/Pi before being
processed in Vectashield mounting medium (Vector Laboratories). The
distribution of fluorescence was analyzed on a Zeiss Axiophot
fluorescence microscope or on a Leica DM IRBE inverted fluorescence microscope.
Flow Cytometry--
To analyze the internalization of
fluorochrome-labeled Tat peptides by FACS, exponentially growing HeLa
cells were dissociated with nonenzymatic cell dissociation medium,
centrifuged at 500 × g, and resuspended in Opti-MEM.
5 × 105 HeLa cells in 250 µl of Opti-MEM were then
incubated with the peptides at the concentration indicated in the
Figure legends. For analysis of Jurkat cells, we used a slightly
different procedure. After 40 min incubation in 1 ml of serum-free
medium, 2 × 106 cells were resuspended in 200 µl of
serum-free medium containing the Tat peptide at 10 µM
concentration. After different times of incubation at 37 °C or
4 °C in the presence of the peptide, the cell suspension was either
centrifuged at 1,300 × g or diluted in 2 ml of
NaCl/Pi and centrifuged at 800 × g. The
cell pellet was washed twice before incubation with trypsin (1 mg/ml)
during 15 min at 37 °C. Cells were then washed once more with
NaCl/Pi and finally resuspended in 500 µl of NaCl/Pi
containing 0.1 mM propidium iodide (Molecular Probes).
In some experiments, cells were incubated with 10 mM sodium
azide in the presence of 6 mM 2-deoxy-D-glucose
for 1 h to deplete cellular ATP.
Fluorescence analysis for both cell lines was performed with a FACScan
fluorescence-activated cell sorter (BD Biosciences). Cells stained with
propidium iodide were excluded from further analysis. A minimum of
20,000 events per sample was analyzed.
Cellular Uptake of Tat and (Arg)9 Peptides--
The
distribution of fluorescein- or Alexa 488-tagged Tat peptide (green
fluorescence) and that of Alexa 546-conjugated transferrin or FM 4-64 (red fluorescence) were followed in living unfixed HeLa and CHO cells
(Fig. 1). On both cell lines, a punctate
cytoplasmic distribution of the peptide was observed (Fig. 1,
A and E) similar to the localization of the
endocytic markers used in this study (Fig. 1, B and
F). On both cell lines, the Tat peptide predominantly co-localized with the marker of endocytosis (transferrin or FM 4-64)
(Molecular Probes) in living cells as indicated by the resulting yellow
coloration (Fig. 1, C and G). However, a mild
fixation with formaldehyde drastically changed the distribution of the Tat peptide (giving rise to the characteristic nuclear localization reported in most published data), whereas the distribution of transferrin or FM 4-64 remained unchanged (Fig. 1, D and
H). These data indicate that even mild fixation led to
artifactual redistribution of Tat peptide, resulting in its apparent
nuclear localization. Likewise, fluorescein-tagged (Arg)9
appeared to be concentrated in nuclei in formaldehyde-fixed cells,
whereas they were located in cytoplasmic vesicles in unfixed cells
(data not shown).
These images of Tat localization in living cells suggest that
endocytosis plays an important role in the uptake of the CPPs at
variance with the prevailing concept and prompted us to re-evaluate their uptake mechanism.
Flow Cytometry Analysis of Tat and (Arg)9 Peptide
Uptake--
FACS analysis is a conventional tool to quantify cellular
association of fluorochrome-tagged peptides. However, because flow cytometry does not discriminate between membrane-bound and internalized fluorochrome, special care should be taken to minimize any contribution of surface-bound peptide in measuring peptide uptake. A simple and
versatile protocol involving trypsin treatment of the cells before FACS
analysis has been used here to assess cellular uptake of
fluorochrome-labeled Tat and (Arg)9 peptides. We incubated cells with fluorescein-labeled peptides at 37 °C or 4 °C for
various periods of time. The trypsinization step was either included or omitted in the washing protocol, and cell fluorescence was measured by
flow cytometry. As shown in Fig. 2 for
HeLa and Jurkat cells incubated with Tat or (Arg)9, FACS
analysis in the absence of trypsin treatment generally overestimated
the cell uptake. At 4 °C, most of the signal was trypsin-sensitive
(Fig. 2A versus 2B and Fig.
2C versus 2D). For a given
temperature, the extent of the overestimation varied according to cell
line and peptide. These data suggest that trypsin treatment removed
surface-bound peptide by digestion of peptide and/or membrane proteins,
whereas simple washing with NaCl/Pi did not remove all
bound peptide. This is not very surprising, considering the highly
cationic nature of these peptides and the overall negative charge of
the cell surface. A standard protocol including a trypsin treatment
before FACS analysis was therefore used in all subsequent
experiments.
An intriguing feature of CPP behavior was their reported fast
internalization. We measured the kinetics of CPP uptake using flow
cytometry as cell-associated fluorescence after the trypsin digestion
step. Accumulation of Tat in Jurkat cells (Fig.
3A) was slow, with kinetics
comparable to that of the classical marker of endocytosis FM 4-64 (Fig.
3B). Data in Fig. 3 also show that FM 4-64 endocytosis (Fig.
3B) and Tat uptake (Fig. 3A) were both severely
impaired by incubation of cells at 4 °C. This is in agreement with
an energy-dependent mechanism of internalization and at
variance with early data in the field. Again, similar data have been
obtained in HeLa and in CHO cells (data not shown).
An energy-dependent uptake mechanism was also confirmed in
the experiments in which the cellular ATP pool was depleted by preincubation of the cells with sodium azide and deoxyglucose. ATP
depletion significantly reduced the uptake of Tat (Fig.
4A) and of (Arg)9
(Fig. 4B) in HeLa cells. The extent of the inhibition was
close to that observed for transferrin, a classical marker of
receptor-mediated endocytosis (data not shown). Similar results were
obtained for Jurkat cells (data not shown).
Cell Uptake and Intracellular Distribution of Tat-PNA
Conjugates--
CPPs are being considered as promising new tools for
the delivery of non-permeant or poorly permeant drugs. In this work, we
tested Tat peptide conjugated to peptide nucleic acids. PNAs are
nuclease-resistant DNA mimics, which hybridize specifically (by
Watson-Crick base pairing) and with unusually high affinity to
complementary RNA. However, the cellular uptake of these uncharged compounds is poor and, unlike DNA, cannot be increased by complexation with cationic lipids, thus limiting their use in antisense strategies (see Ref. 29 for a review). Tat-PNA conjugates carrying a fluorochrome on the N terminus of the Tat (data not shown) or PNA-Tat conjugates carrying a fluorochrome on the N terminus of the PNA (Fig.
5) moiety were taken up in HeLa cells and
accumulated in vesicles. As for Tat peptide, cellular uptake of these
Tat-PNA conjugates measured by flow cytometry was greatly reduced by
incubation at low temperature and by ATP depletion (Fig.
6).
It has been previously reported that cell incubation with various
CPPs results in their rapid uptake with a predominantly nuclear
localization and the lack of the punctate cytoplasmic labeling
characteristic of endocytic uptake (7, 20-22, 30). Intriguingly, the
same picture was observed at 37 °C and at 4 °C or in the presence
of a number of inhibitors of endocytosis and cell metabolism (see ref.
31 for a comprehensive overview). In the present study we found that,
at least for Tat and (Arg)9, even the reportedly mild
cell-fixation protocols commonly used to evaluate uptake by
fluorescence microscopy (32) caused an artifactual redistribution of
these peptides into the nucleus and gave rise to altered images as
compared with the experiments made on unfixed cells. This was not
observed for classical endocytic markers such as transferrin and FM
4-64. This artifact could be related to the highly cationic nature of
the peptides, which is a common and even necessary characteristic for
CPP (21, 23, 24, 33). A high density of positive charges leads to
rather strong binding of the peptides to the overall negatively charged plasma membrane as was evidenced by our flow cytometry analysis data.
Even mild fixation caused a disruption of membrane barrier function and
increased transfer of hydrophilic probes such as propidium iodide into
the cytoplasm and nucleus, as detected by the labeling of the cell
nucleus under these conditions (data not shown). High positive charge
of the peptides most probably causes their strong binding to nucleic
acids and ultimately leads to concentration of CPP into the nucleus,
which is an abundant reservoir of cellular nucleic acids. Cell fixation
should therefore be avoided for further studies on the intracellular
distribution of CPP and of their conjugates to various cargoes. The
cargo itself might influence the final distribution of the conjugate,
as attested to by two recent papers. PNA-Tat (or Antennapedia)
conjugates were found predominantly segregated in endocytic vesicles
(29), in keeping with our own data, whereas phosphorothioate antisense oligonucleotides conjugated to these CPP were at least partly found in
nuclei (12).
Strong binding of CPP to the cell surface also leads to the
overestimation of uptake by flow cytometry, and special care should be
taken to remove membrane-bound peptide for the evaluation of cell
uptake. For flow cytometry experiments, we used a simple protocol based
on the treatment of cells with trypsin before FACS analysis. Although
only trypsin treatment was used in this study, the contribution of the
bound peptide could possibly be eliminated by other means, including
treatment with other proteases. Most probably, trypsin digested the
peptide and decreased binding of the remaining degradation products to
the cell surface. Alternative approaches have recently been proposed
that rely on the use of fluorescence quenching groups. A
3-nitrotyrosine quenching group has been introduced in Transportan (and
in other CPPs), and these modified peptide carriers were conjugated
through a disulfide bridge to a small peptide cargo carrying the
fluorochrome. This elegant approach allows measurement only of cellular
uptake of the peptide-conjugated cargo, because release of the
fluorescent cargo strictly requires the reduction of the disulfide
bridge, which is supposed to occur only in the intracellular reductive environment (34). Along the same lines, Drin et al. (35)
conjugated the Antennapedia peptide to
7-nitrobenz-2-oxo-1,3-diazol-4-yl (NBD), whose fluorescence could be
quenched by dithionite, a membrane-impermeant compound. Cell
association could therefore be discriminated from cell uptake. It is
worth noting that both studies led to the conclusion of a relatively
slow rate of uptake of these CPPs (see below). In contrast to
the trypsin treatment, these interesting strategies require the
synthesis of the appropriate peptide analogs.
Reevaluation of the mechanism of the internalization of Tat and
(Arg)9 peptides demonstrated that, at variance with most
published data, internalization of these peptides was strongly
inhibited by low temperature or by depletion of the cellular pool of
ATP. Moreover, the kinetics of peptide uptake was as slow as that
characteristic of endocytosis. The observation by fluorescence
microscopy of living cells incubated with these peptides or their PNA
conjugates demonstrated a punctate distribution characteristic of
endocytosis and overlapping with common endocytic markers. These
results strongly support the involvement of endocytosis as the major
route for the internalization of Tat and (Arg)9. The
applicability of this conclusion to other CPP with different cargoes
remains to be verified. Also, we cannot formally exclude the
possibility that a small fraction of CPP enters cells by an
endocytosis-independent but biologically relevant pathway.
One can hypothesize that the high positive charge of the peptides used
in the present study promote binding of the peptide and of the
conjugated cargo to the cell surface. Interestingly, the
internalization of the full-length HIV-1 Tat protein required an
interaction of the basic domain of full-length Tat with cell surface
heparan sulfate proteoglycans (36, 37). Whether these receptors or
other cell surface determinants are involved in the endocytosis of CPP
will have to be reinvestigated using the tools described here.
In conclusion, we present here data unveiling artifacts associated with
the experimental techniques most commonly used to study the
internalization of CPP. Using simple and versatile improved methodologies, we reestablish the role of endocytosis in the
internalization of cationic CPP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C until further use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cell fixation affects intracellular
distribution of Tat peptide. HeLa (panels
A-D) or CHO (panels E-H) cells
were incubated with the 1 µM or 10 µM
flurochrome-tagged Tat peptide respectively, with 25 µg/ml
transferrin, or with 10 µM FM 4-64 for 15 min as detailed
under "Materials and Methods." Cells were either fixed with
formaldehyde before fluorescence microscopy or left untreated.
A, Alexa 488-tagged Tat peptide in unfixed HeLa cells
(green fluorescence); B, Alexa
546-tagged transferrin in unfixed HeLa cells (red
fluorescence); C, co-localization of Tat peptide
and transferrin incubated together in unfixed cells (yellow
fluorescence); D, nuclear localization of Tat
peptide (green fluorescence) and vesicular
localization of transferrin (red fluorescence) in
fixed cells; E, fluorescein-tagged Tat peptide in unfixed
CHO cells (green fluorescence); F, FM
4-64 in unfixed CHO cells (red fluorescence);
G, co-localization of Tat peptide and FM 4-64 incubated
together in unfixed CHO cells; H, nuclear localization of
Tat peptide (green fluorescence) and vesicular
localization of FM 4-64 (red fluorescence) in
fixed CHO cells.
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Fig. 2.
Cell-associated Tat and
(Arg)9 with and without trypsin
treatment. HeLa cells were incubated for 15 min in the presence of
1 µM fluorescein-tagged (Arg)9 at 37 °C
(A) or 4 °C (B) with (red
curves) or without (blue curves)
trypsin treatment before FACS analysis. Jurkat cells were incubated for
15 min in the presence of 10 µM fluorescein-tagged Tat at
37 °C (C) or 4 °C (D) with (red
curves) or without (blue curves)
trypsin treatment before FACS analysis. The black
curves correspond to cells incubated in the absence of
peptides.
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Fig. 3.
Kinetics of cell uptake of Tat at 37 °C
and 4 °C. Jurkat cells were incubated in the presence of 10 µM fluorescein-tagged Tat (A) or 10 µM FM 4-64 (B) at 37 °C (closed
circles) or at 4 °C (squares) for the
indicated periods of time. Samples were treated with trypsin before
FACS analysis.
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Fig. 4.
Depletion of cellular ATP inhibits uptake of
Tat and (Arg)9. HeLa cells were incubated for 15 min in the presence of 5 µM fluorescein-tagged Tat
(A) or 2 µM fluorescein-tagged
(Arg)9 (B). Intracellular ATP pool has been
depleted (blue curves) or not (red
curves) by preincubation with sodium azide and deoxyglucose.
Samples were treated with trypsin before FACS analysis.
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Fig. 5.
Intracellular distribution of Tat-PNA
conjugates. HeLa cells were co-incubated for 15 min with 1 µM fluorescein-tagged Tat-PNA (green
fluorescence) (A) and 25 µg/ml Alexa 546-tagged
transferrin (red fluorescence) (B);
C, image merging of Tat-PNA peptide and transferrin
(yellow fluorescence). Cells were not fixed
before observation by fluorescence microscopy.
View larger version (12K):
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Fig. 6.
Cellular uptake of Tat-PNA is inhibited at
4 °C and by depletion of cellular ATP. HeLa cells were
incubated for 15 min in the presence of 1 µM
fluorescein-tagged Tat-PNA. A, cells were incubated at
37 °C (red curve) or at 4 °C
(blue curve). B, cells were
preincubated (blue curve) or not (red
curve) with sodium azide and deoxyglucose to deplete
cellular ATP. Samples were treated with trypsin before FACS
analysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank C. Rispal for taking part in FACS analysis, P. Travo for assistance in fluorescence microscopy, and E. Leikina, A. Mittal, and P. Rabinovich for helpful discussions.
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FOOTNOTES |
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* This work was supported by grants from Association pour la Recherche sur le Cancer, Ligue Nationale Francaise de Recherche contre le Cancer, and Groupement des Entreprises Francaises de Lutte contre le Cancer (to B. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: NICHD, National
Institutes of Health, 10 Center Dr., Bldg. 10, Rm. 10D05, Bethesda, MD
20892. Tel.: 301-402-9010; Fax: 301-594-0813; E-mail:
melikovk@mail.nih.gov.
Published, JBC Papers in Press, October 30, 2002, DOI 10.1074/jbc.M209548200
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ABBREVIATIONS |
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The abbreviations used are: CPP, cell-penetrating peptides; FACS, fluorescence-activated cell sorter; PNA, peptide nucleic acid; Fmoc, N-(9-fluorenyl)methoxycarbonyl; CHO, Chinese hamster ovary; Bhoc, benzhydryloxycarbonyl; PyBOP, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate; DIPEA, diisopropylethylamine; PyAOP, 7-azabenzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; DIPEA, diisopropylethylamine; HATU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate.
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