1. A Novel Cell-penetrating Peptide, M918, for Efficient Delivery of Proteins and Peptide Nucleic Acids 
Samir El-Andaloussi, Henrik J Johansson, Tina Holm, Ülo Langel 
Molecular Therapy 
Volume 15, Issue 10, Pages (October 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
M918 efficiently translocates into various cells in a non-toxic fashion. (a) One day before the experiment,200,000 cells/well were seeded in 12-well plates. HeLa cells were treated for 1 hour with 5 μmol/l fluoresceinyl-labeled M918, Pen, or TP10, in serum-free Dulbecco's modified Eagle's medium to a final volume of 500 μl. Cells were washed in HEPES Krebs Ringer, trypsinized, and centrifuged at 1,000g. Pellets were lysed in 0.1 mol/l NaOH, and fluorescence was measured at 494/518 nm. Fluorescence was recalculated to the amount of internalized peptide per milligram of protein. As a negative control of cellular uptake, YDEGE peptide was used. (b) Uptake in additional cell lines was performed in the same way as in a. (c) Membrane leakage was measured by lactate dehydrogenase (LDH) release 30 minutes after peptide treatment. TP10 (25 μmol/l) represents the positive control. (d) Long-term toxicity was assessed by wst-1 after exposing cells twice to new peptide, using the indicated concentrations, after 24 hours and 48 hours, measured after 72 hours. Staurosporine (1 μmol/l) is used as a positive control. The values represent the mean of at least three independent experiments performed in triplicate (mean ± SEM, n = 3). CHO, Chinese hamster ovary; VEGF, vascular endothelial growth factor.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Confocal microscopy of M918 displays mainly vesicular localization inside cells. (a) MCF-7, (b) Hifko, and (c) HeLa cells were seeded in NUNC 8 chambers 2 days before the experiment to reach 60% confluence. After 30 minutes' treatment with 1 μmol/l fluoresceinyl-labeled M918 peptide in HEPES Krebs Ringer (HKR), cells were washed three times with HKR before analysis with confocal microscopy.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
CPPs promote protein transduction. HeLa cells (200,000 cells/well) were seeded in 12-well plates 1 day before treatment. (a, b) Streptavidin (SA) or (c, d) avidin (0.2 μmol/l) was either (a, c) incubated for 30 minutes in 50 μl 0.9% NaCl with 5 μmol/l peptide or (b, d) incubated with 1 μmol/l biotin-labeled peptide enabling formation of four nearly irreversible bonds to proteins, and added to HeLa cells for 90 minutes in 500 μl serum-free Dulbecco's modified Eagle's medium. Cells were then treated as in Figure 1. The values represent the mean of at least three independent experiments performed in triplicate (mean ± SEM, n = 3). FITC, fluorescein isothiocyanate.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
M918 internalizes to cells via endocytosis independent of glycosaminoglycans on the cell surface. (a) HeLa cells (200,000 cells/well) were seeded in 12-well plates 24 hours before the experiment and were incubated with 5 μmol/l fluoresceinyl-labeled M918 or 25 μg/ml rhodamine-labeled transferrin for 1 hour in 500 μl serum-free Dulbecco's modified Eagle's medium and pre-treated for 30 minutes with 75 μmol/l chloroquine (cq), 0.2 mol/l sucrose, or low temperature. Cells were then treated as in Figure 1. (b) Chinese hamster ovary (CHO) and CHO2242 cells treated as in a. Uptake of the three peptides in CHO cells was set to 100%. The values represent the mean of at least three independent experiments performed in triplicate (mean ± SEM, n = 3). *P < 0.05, **P < 0.01, ***P < (a) Analysis of variance Dunnett's, (b) t-test.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
M918 partially co-localizes with both transferrin and dextran in MCF-7 cells. (a) Brightfield image, (b) M918, (c) endocytosis markers, dextran or transferrin, (d) composite. MCF-7 cells (20,000) seeded 2 days before the experiment in NUNC 8 chambers were treated with 1 μmol/l fluoresceinyl-labeled peptide for 30 minutes together with 1 mg/ml of the fluid-phase endocytosis marker rhodamine-labeled dextran or 25 μg/ml of the clathrin-mediated endocytosis marker rhodamine-labeled transferrin. Cells were washed three times with HEPES Krebs Ringer before analysis by confocal microscopy.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
M918-PNA enters cells via macropinocytosis and dose-dependently induces splice correction. HeLa pLuc 705 cells (60,000 cells/well) were seeded 48 hours before the experiment in 24-well plates, after which cells were treated with peptide–PNA conjugates for 1 hour in serum-free Dulbecco's modified Eagle's medium followed by 16 hours in serum medium. (a) Splice correction after treatment with 5 μmol/l peptide–PNA conjugates presented as fold increase in splicing over untreated cells. The controls for specificity and delivery efficacy of M918 are 10 μmol/l inverted PNA (invPNA) and PNA, respectively. (b) Uptake and splicing after treatment with different M918-PNA concentrations presented as relative light units (RLU)/mg. (c) Cells were pre-treated with 4 μmol/l cytochalasin D, 50 nmol/l wortmannin, or pre-maintained at 4 °C for 30 minutes and treated at for 1 hour with 5 μmol/l M918-PNA. RLU/mg values obtained with conjugate only were set to 100% splice correction. (d) Cells were treated as in a with M918-PNA at the indicated concentrations together with 75 μmol/l chloroquine (cq). The values represent the mean of three independent experiments each performed in triplicate (mean ± SEM, n = 3). *P < 0.05, **P < 0.01, ***P < (c) Analysis of variance (ANOVA) Dunnett's, (d) ANOVA Bonferroni. PNA, peptide nucleic acid.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions