Effect of Dd and Dd-BLM conjugate on HeLa cells.

<p>(A) Flow cytometry analysis of Dd (green curve) and Dd-BLM (pink curve) cell entry. Cells were treated with appropriate vector for 1 h at 37°C as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>. The blue curve shows the antibody background in the absence of Dd. (B) Cells were treated with 1 µg Dd or Dd-BLM for indicated times and analyzed with anti-Dd serum (in red) by confocal microscopy, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>. Nuclei were stained blue with DAPI. Last row shows the 50 h-treatment without nuclear staining. Scale bar equals 20 µm.</p

Cell toxicity of bleomycin (BLM) delivered with the aid of Dd.

<p>BLM was chemically attached to Dds as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>. (A) Characterization of Dd-BLM conjugate by mass spectrometry analysis (Maldi). (B) DLS analysis of the BLM conjugate. (C) MTT assay of cell toxicity. HeLa cells were treated with free BLM (0.13 µM), Dd (1 µg) and Dd-BLM (1 µg delivering 0.08 µM BLM), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>.</p

Kinetics of ds DNA breaks as jugded by induction of γ-H2AX foci.

<p>HeLa cells were treated either with Dd, with free BLM or Dd-BLM for indicated periods and analyzed with anti-γ-H2AX Ab (in red) and with anti-tubulin Ab (in green) by confocal microscopy, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>. Scale bar equals 10 µm.</p

Purification of recombinant Ad3 DB expressed in the baculovirus system.

<p>(A) Dodecahedra initially purified on sucrose density gradient were fractionated on a Q-Sepharose column in 20 mM Tris buffer, pH 7.5, using NaCl gradient. (B) Analysis of purified Dds. Left panel - negative stain electron microscopy (EM) of Dds purified on sucrose density gradient. Middle and right panels - non-denaturing agarose gel electrophoresis of fractions recovered from the Q-Sepharose column with detection with ethidium bromide (EtBr, middle panel) followed by Commassie Brilliant Blue (CBB) staining (right panel). (C) Negative stain EM showing free pentameric bases recovered in peak 1 (left panel) and complete dodecahedra in peak 2 (right panel) of the Q-Sepharose column (P1 and P2 in A). Scale bar equals 100 nm. (D) Flow cytometry analysis of HeLa cells transduced with Dd (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>). Sucrose density gradient-purified Dds – purple curve, Q-Sepharose-purified Dds - green curve. Blue curve shows the antibody background in the absence of Dd.</p

Dd stability upon lyophilization, inside HeLa cells and in human serum.

<p>(A) Purified Dds were dialyzed overnight at 4°C against water or 150 mM (NH₄)₂SO₄ in water. Mannitol (0.4%) and sucrose (0.4%) were added to samples marked „Cryoprotect. +”. Dd samples were frozen at −80°C, dried in speed-vac or lyophilized (marked Lyoph. +). Dry samples were reconstituted in the starting volume of water. All preparations were centrifuged for 30 min at 13000 rpm and the supernatants were applied onto native agarose gel. (B) Stability of Dd after application to HeLa cells. Purified Dds (2 µg in 100 µl) were applied to 2×10⁴ portions of HeLa cells. After indicated periods of penetration cell lysates were analyzed on SDS-PAGE (left panel) or on native agarose gel (two right panels). Control Dd samples contained 30 ng protein, while control Pb sample contained 10 ng protein. (C) Stability of Dd upon incubation in human serum. Samples of Dd concentrated by ultrafiltration in Microcon unit (Millipore) (5 µg each) were incubated in human serum (HS) at 4°C for 2 h (lane 4) and at 37°C for 15 min or for 2 h (lanes 5 and 6, respectively). Samples were resolved by native agarose gel electrophoresis and analyzed by Western blot performed with anti-Dd serum. The upper part shows CBB stained gel with proteins remaining after transfer, and the lower part the developed Western blot. Lanes 1 and 7 show Dd non treated or incubated for 2 h at 37°C, respectively, in the absence of serum. Lanes 2 and 3 show human serum after 2 h incubation at 4 and 37°C, respectively. Dd samples incubated with the serum are denoted in bold.</p

Thermal Dd stability as a function of pH and ionic strength.

<p>(A) Dd was analyzed by dynamic light scattering (DLS) at different pH in the presence of 150 mM NaCl as a function of temperature, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>. (B) Native gel analysis of Dds and Pbs, in CAPS pH 9, and carbonate buffer, pH 10. Some samples were subjected to temperature treatment imitating DLS temperature gradient (marked DLS). (C) DLS analysis carried out on Dds in PBS under different ionic strength conditions. Mean values of three apparatus readings are shown.</p

Dd stability under different conditions of temperature, pH and ionic strength, analyzed by native gel electrophoresis.

<p>Purified Dds were dialyzed overnight at 4°C (A, B) or for 24 h at 37°C (C) against different buffers at the indicated pH. (A) Effect of pH on Dd solubility. Dd was dialyzed against the following 50 mM buffers: MES, pH 6; Hepes, pH 7; Tris, pH 8 and CAPS, pH 9. Left panel: samples were prepared in duplicates and after dialysis the second batch was centrifuged at 13000 rpm for 30 min. The first four lanes contain the dialyzed samples; the next lanes contain the supernatants after centrifugation. Right panel: samples were dialyzed against the same buffers as described for the left panel and also against citric acid, pH 4, and acetic acid, pH 5, but all buffers contained 150 mM NaCl. Samples were prepared in duplicates and one batch was kept at 4°C while the second one was incubated for 20 min at 30°C. Soluble proteins contained in the supernatants obtained by 30 min centrifugation at 13000 rpm were applied on agarose gel. (B) Dd stability in carbonate buffer. Carbonate buffer (100 mM) was prepared at the indicated pH and used for Dds dialysis. Some samples were incubated at 37°C for 20 min. All samples were centrifuged as above and the supernatants were electrophoresed on native agarose gel. First and last lanes, control Dd and Pb preparations, respectively. (C) Effect of ionic conditions on Dd thermal stability. Dds were dialyzed for 24 h at 37°C against the purification buffer containing NaCl or (NH₄)₂SO₄ (AS) at indicated concentrations or against PBS, pH 7.5. Non-treated samples (T for total) or supernatants after centrifugation at 13000 rpm for 30 min (S) were analyzed on the agarose gel. The first two lanes contain control Dd while the last lane contains Pb preparations, all in purification buffer.</p

Interaction of Dd with WW protein.

<p>(<b>A</b>) Aliquots of serially diluted proteins applied in duplicate to a nitrocellulose membrane were overlaid with GST-WW protein as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046075#s4" target="_blank">Materials and Methods</a>. Interacting GST-WW protein was detected with anti-GST-HRP antibody. The average amount of interacting WW protein was determined by densitometry. (<b>B</b>) Cartoon of the trefoil opening between three Pb pentamers shown in pastel colors. A WW domain (pdb 2jo9 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046075#pone.0046075-Yamasaki1" target="_blank">[38]</a>) in green is shown bound to the ¹⁹PPxY motif of the disordered N-terminus (residues 1–47) shown in red. (<b>C</b>) The same model viewed from the side represented with a transparent surface. The N-terminus (residues 1–47) is shown in red and the WW domain in green, both as space-filling models. Residues 22–47 can only be seen thanks to the transparent surface of the Pbs. The model does not involve any precise docking but permits visualizing possible positions and relative sizes of the involved molecules.</p

Ad3 dodecahedron structure.

<p>(<b>A</b>) The secondary structure of the Ad3 Pb in the orthorhombic crystal form is shown together with the one of Ad2 (pdb entry 1×9p <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046075#pone.0046075-Zubieta1" target="_blank">[13]</a>). The corrected sequence (GenBank acc. no ABB17799.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046075#pone.0046075-Sirena1" target="_blank">[26]</a>) of the Ad3 Pb with ⁵⁸SELS instead of ⁵⁸SDVS has been used. Disordered or proteolysed residues at the N-terminus which are invisible in the structure are indicated with a black bar. PPxY motifs are marked in yellow. Glu451 involved in a putative Ca²⁺ binding site is highlighted with a green background, the variable loop with a pink background, the RGD loop with a light blue background. The peptide of the RGD binding loop that is probably missing due to proteolysis, is marked with a gray bar. A dark blue background marks contact residues involved in the Dd formation. Secondary structure elements are labeled as in Zubieta <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046075#pone.0046075-Zubieta1" target="_blank">[13]</a> whenever possible. (<b>B</b>) Cartoon representation of the overall structure of an Ad3 Pb in the cubic crystal form. One of the Pb subunits is highlighted in red. The dotted line symbolizes the part of the RGD loop, which is invisible in electron density. The black arrow points to the putative calcium ion. The ‘dangling’ N-terminal ends at the bottom are in fact involved in strand-swapping with other Pbs. (<b>C</b>) Alignment of the Ad3 Pb monomer structure (magenta) from the orthorhombic crystal form in presence of fiber peptide (orange) with the one of Ad2 (cyan).</p

Analysis of dodecahedra formation by Ad3 Pb mutants.

<p>(<b>A</b>) Amino acid sequence of Pb protein regions responsible for Dd integrity. (<b>B</b>) Structure of regions 2 and 3 involved in Dd formation. These residues are located around the 2-fold axes of the dodecahedron. The light and dark green molecules belong to one Pb, the magenta and orange molecules to another one. Dotted lines mark potential hydrogen bonds, although they cannot be identified unambiguously due to the limited resolution. (<b>C</b>) Sucrose density gradients of rwtDd and mutants. Cell extracts obtained from expressing HF cells were fractionated and analyzed on denaturing PAGE as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046075#s4" target="_blank">Material and Methods</a>. (<b>D</b>) Native gel analysis of Q-Sepharose fractions eluted with 370 mM NaCl and revealed with CBB stain. Pb and wt Dd were used as internal standards. Note the shift of Dd bands due to changed net charges of Dd constructs. (<b>E</b>) Electron microscopy of wt and mutant Dd. Samples shown in <b>C</b> were analyzed after dialysis.</p