Displayed peptide YTYDPWLIFPAN provided penetration of phage particles into MDA-MB-231 cells.

<p>(А) Fluorescence microscopy of MDA-MB-231 cells incubated with phage clone 1.1 and control bacteriophage at 4°C. (B) Fluorescence microscopy and (C) confocal fluorescence microscopy of MDA-MB-231 cells incubated with phage clone 1.1 and control bacteriophage at 37°C and further washes from bacteriophages bound to the surface. Fluorescence microscopy was performed using mouse anti-М13 and Alexa Fluor 488 donkey anti-mouse IgG (H+L) antibodies. For visualization of nuclei, cells were stained with DAPI. (D) Electron microscopy of MDA-MB-231 cell after 1 h of incubation with phage clone 1.1 at 37°C; ultrathin section. The arrow points to a phage particle.</p

Enhanced cytotoxic outcome in combination of RL2 with CQ.

<p>MDA-MB-231 cells were used as a model. <b>A</b>. Dose-dependence of CQ cytotoxicity was calculated using MTT data of three independent experiments. Shown are mean values ± SD. <b>B</b>. Cells were treated with different concentrations of RL2 in the presence or absence of CQ, and after 48 h cell viability was measured by MTT assay. <b>C</b>. Cells were incubated with RL2 (0.2 mg/mL), CQ (5 μM) or a combination for 24 h and the percentage of cells with active caspase -3,-7 was analysed using flow cytometry. Shown are mean values ± SD of three independent experiments. The asterisks indicate significant difference from control (*, p<0.05). <b>D</b>. Cells were incubated with RL2 (0.2 mg/mL), CQ (5 μM) or a combination for 24–48 h. Cathepsin D activity was analysed using a fluorescence substrate (labeled with methyl cumaryl amide) by fluorimetry as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093921#s2" target="_blank">Materials and methods</a>’ section. RFU – relative fluorescence units per microgram protein of sample. Shown are mean values ± SD of three independent experiments. The asterisks indicate significant difference between indicated groups (**, p<0.05), ns - not significant difference between groups (p>0.05).</p

Relative binding of the selected bacteriophage clones to cells.

<p><b>Displayed peptides 1.1 –</b>YTYDPWLIFPAN, <b>1.2 –</b>FIPFDPMSMRWE <b>and 1.3 –</b>SLPVYAPALTSR. (A) MDA-MB-231, MCF-7 and BN-2 cells were incubated with selected phage clones and control bacteriophage. After incubation and a series of washings, the titer of bacteriophages bound to cells was determined. Cells of untransformed human breast BN-2 primary culture were used as a negative control. The value for the binding of the control bacteriophage was used as a normalization value. Data are presented as mean ± SD. Data were statistically analyzed using one-way ANOVA with post hoc Fisher test. A p value < 0.05 was considered to be statistically significant. (B) Flow cytometry of MDA-MB-231, MCF-7 and BN-2 cells incubated with clone 1.1 and control bacteriophage. Flow cytometry was performed using mouse anti-М13 and Alexa Fluor 488 donkey anti-mouse IgG (H+L) antibodies.</p

RL2 regulates activity of apoptosis-related genes.

<p><b>A.</b> MDA-MB-231 cells were treated with RL2 (0.2 mg/mL) for the indicated time followed by total RNA isolation. Levels of transcripts were analysed by real time RT PCR with specific primers and were presented as relative values normalized to the level of GAPDH mRNA. n = 3; the error bars represent standard deviations. The asterisks indicate significant difference from control (0 h), *p<0.05, or between indicated groups, **p<0.05, ns - not significant difference between groups (p>0.05). <b>B</b>. MDA-MB-231 cells were treated with RL2 (0.2 mg/mL) followed by preparation of whole-cell lysates, which were then subjected to Western blot with the indicated antibodies. Tubulin was used as the internal control. Results shown are representative of five independent experiments.</p

Separation of proteins of RL2-bound fraction.

<p>Coomassie stained gel. RL2-bound fractions of MCF-7 lysates were prepared as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093921#s2" target="_blank">Materials and methods</a>’ section and then were separated by 15% SDS-PAGE gel. Bands indicated (*) were excised followed by in-gel trypsin digestion for mass spectrum analysis. Other bands were not investigated. Shown is representative gel of two independent experiments. Lane 1 – fraction eluted from non-modified Sepharose (control); Lane 2 – fraction eluted from RL2-modified Sepharose with 300 mM NaCl, flow rate was 1 mL/min and detection wavelength was 280 nm. M – molecular weights marker (14.4–116.0 kDa).</p

Fusion proteins enhanced cytotoxic outcome and delayed tumor development.

<p>(A) MDA-MB-231 and MCF-7 cells were treated with different concentrations of RL2, fusion proteins or saline (control) for 48 h and MTT analysis was performed. Tumor cell viability was determined relative to the viability of the control cells (incubated without the proteins). Data are presented as mean ± SD. (B) SCID mice with subcutaneously grafted MDA-MB-231 tumors, which reached an average (in the group) size of 20 ± 10 mm³, were subjected to intravenous injection of fusion proteins (RL2, T3-RL2, RL2-iRGD or RL-iRGD-His) at a dose of 40 mg/kg in saline three times every second day. The control group of mice received saline with the same mode of as administration the proteins. Tumors were excised and weighed on the 17th day after the last injection. Boxes represent the 25th, 50th and 75th percentiles. Squares with lines represent the median. Whiskers represent minimum/maximum. Data were statistically analyzed using one-way ANOVA with post hoc Fisher test; a p value < 0.05 was considered to be statistically significant.</p

Displayed peptide YTYDPWLIFPAN provided penetration of phage particles into MDA-MB-231 cells.

<p>(А) Fluorescence microscopy of MDA-MB-231 cells incubated with phage clone 1.1 and control bacteriophage at 4°C. (B) Fluorescence microscopy and (C) confocal fluorescence microscopy of MDA-MB-231 cells incubated with phage clone 1.1 and control bacteriophage at 37°C and further washes from bacteriophages bound to the surface. Fluorescence microscopy was performed using mouse anti-М13 and Alexa Fluor 488 donkey anti-mouse IgG (H+L) antibodies. For visualization of nuclei, cells were stained with DAPI. (D) Electron microscopy of MDA-MB-231 cell after 1 h of incubation with phage clone 1.1 at 37°C; ultrathin section. The arrow points to a phage particle.</p

Sequences and frequencies of the peptides displayed by bacteriophages selected after the third (30 clones) and fourth (30 clones) rounds of biopanning on MDA-MB-231 cancer cells.

<p>Sequences and frequencies of the peptides displayed by bacteriophages selected after the third (30 clones) and fourth (30 clones) rounds of biopanning on MDA-MB-231 cancer cells.</p

Identification of RL2-interacting proteins A and B.

<p>Protein identification was based on the SwissProt database using MALDI spectra of protein typsin lysates.</p

RL2 treatment induces autophagic changes in MDA-MB-231 cells.

<p><b>A.</b> Cells were treated with RL2 (0.2 mg/mL) or non-treated (control) for 8 h and fixed with 4% paraformaldehyde followed by immunostaining with anti-LC3A/B primary and FITC-conjugated secondary antibody. Images are representative of two independent experiments. <b>B</b>. MDA-MB-231 cells were treated with RL2 (0.2 mg/mL) for various time points (0, 2, 6 and 24 h). Total lysates were prepared and subjected to Western blot analysis. Shown are representative blots of five independent experiments.</p