QSOX1 function in cell invasion is related to its role in autophagy.

<p>(<b>A</b>) MCF-7 C, QSOX1S-1, QSOX1S-2 and (<b>B</b>) MDA-MB-231 shC, shQSOX1-1, shQSOX1-2 cells were seeded on polycarbonate filters coated with Matrigel and incubated for 24 h, in the presence or absence of autophagy inhibitors 3-MA (10 mM) or wortmannin (100 nM). Inserts were then stained with a 2% crystal violet solution and photographed. A representative image of ten fields of view (FOV) of each membrane is shown. Scale bar represents 30 µm. (<b>C and D</b>) 10 FOV were randomly selected and the number of invasive cells, observed in A and B respectively, was determined. Data are means ± S.D. of two independent experiments performed in duplicate. *P<0.05 compared to the control.</p

QSOX1 regulates the levels of autophagic markers, p62 and LC3-II.

<p>(<b>A</b>) MCF-7 C (lanes 1–3), QSOX1S-1 (lanes 4–6), QSOX1S-2 (lanes 7–9) and (<b>B</b>) MDA-MB-231 shC (lanes 1–3), shQSOX1-2 (lanes 4–6), shQSOX1-1 (lanes 7–9) cells were cultured for 24 h. Cells were then lysed and total proteins (40 µg) were separated on a 12.5% SDS-PAGE followed by immunoblotting using anti-p62, anti-LC3 and anti-actin antibodies. (<b>C and D</b>) p62 and LC3-II levels, observed on the western blot in A and B respectively, were quantified using the Image Lab software. Data are means ± S.D. of one representative experiment performed in triplicate. *P<0.05 compared to the control. (<b>E and F</b>) After reverse transcription, relative p62 mRNA levels in MCF-7 C, QSOX1S-1, QSOX1S-2 (E) and MDA-MB-231 shC, shQSOX1-1, shQSOX1-2 (F) cells were determined by qPCR. H3B-2 mRNA was used for normalization. Data are means ± S.D. of one representative experiment performed in triplicate.</p

QSOX1 is induced following a nutrient stress and protects against cell death.

<p>(<b>A, B</b>) MDA-MB-231 shC cells were cultured in the presence or absence of EBSS for 2, 4, 6 and 8 h. (<b>A</b>) After a reverse transcription step, relative QSOX1 mRNA expression was determined by qPCR. H3B-2 mRNA was used for normalization. Data are means ± S.D. of two independent experiments performed in triplicate. *P<0.05, compared to the control. (<b>B</b>) Cells were lysed and total proteins (50 µg) were separated on a 10% SDS-PAGE followed by immunoblotting using anti-QSOX1 and anti-actin antibodies. QSOX1 levels were quantified using the Image Lab software. NS: Non specific signal. (<b>C, D</b>) MCF-7 C, QSOX1S-1, QSOX1S-2 and MDA-MB-231 shC, shQSOX1-1, shQSOX1-2 cells were cultured in the presence or absence of EBSS for 8 h. Cell viability was estimated using a MTT assay (<b>C</b>) or a trypan blue exclusion assay (<b>D</b>). Results were expressed as a ratio between treated and untreated cells. Data are means ± S.D. of two independent experiments performed in 8 replicates for the MTT assay and in duplicate for the trypan blue exclusion assay). *P<0.05 compared to the control.</p

QSOX1 inhibits autophagosome/lysosome fusion.

<p>(<b>A</b>) MCF-7 C, QSOX1S-1, QSOX1S-2 and (<b>B</b>) MDA-MB-231 shC, shQSOX1-1, shQSOX1-2 cells were transfected with the pGFP-LC3 vector. 24 h after transfection, cells were incubated in complete medium supplemented with 500 nM Lysotracker red for 1 h. Scale bar represents 10 µm. (<b>C and D</b>) Colocalization between Lysotracker-stained acidic vesicles and GFP-LC3-positive autophagosomes, observed in A and B respectively, was quantified using a confocal microscope and the Pearson’s coefficient using coloc_2 plugin (ImageJ software). The data representative of two independent experiments are shown. *P<0.05 compared to the control. Arrows indicate colocalization. (<b>E</b>) MCF-7 C, QSOX1S-1, QSOX1S-2 and (<b>F</b>) MDA-MB-231 shC, shQSOX1-1, shQSOX1-2 cells were transfected with the pGFP-LC3 vector and then immunostained for LAMP1. Arrows indicate colocalization and Scale bar represents 10 µm. (<b>G and H</b>) Colocalization of the autophagosome marker GFP-LC3 and the lysosomal marker LAMP1 was analyzed using a confocal microscope and the Pearson’s coefficient using coloc_2 (ImageJ software). A representative image of two independent experiments is shown. *P<0.05 compared to the control.</p

The extinction of QSOX1 expression in tumors is correlated with low levels of p62.

<p>(<b>A</b>) Tissue sections of MDA-MB-231 shC, shQSOX1-2 and shQSOX1-1 tumors fixed in formol were subjected to p62 immunostaining. Sections were then analyzed by confocal microscopy and a representative image of 3 independent experiments performed in duplicate is shown. Scale bar represents 30 µm. (<b>B</b>) The number of p62 puncta per cell and (<b>C</b>) the number of p62-positive cells were determined using the ImageJ software. To determine the number of p62 puncta, 40 cells per tumor were randomly counted. To determine the number of p62-positive cells count, 23 fields were randomly chosen. *P<0.05 compared to the control.</p

QSOX1 inhibits autophagic flux.

<p>(<b>A</b>) MCF-7 C, QSOX1S-1, QSOX1S-2 and (<b>B</b>) MDA-MB-231 shC, shQSOX1-2, shQSOX1-1 cells were cultured in complete medium with or without 100 nM bafilomycin A1 for 8 h. Cells were then lysed and total proteins (40 µg) were separated on 12.5% SDS-PAGE followed by immunoblotting with anti-p62, anti-LC3 and anti-actin antibodies. p62 and LC3-II levels were quantified using the Image Lab software. (<b>C and D</b>) The autophagic flux, observed in A and B respectively, was determined as the ratio of LC3-II protein levels in the presence of bafilomycin A1 versus the levels in the absence of bafilomycin A1. Data are means ± S.D. of three independent experiments. *P<0.05 compared to the control. (<b>E</b>) MCF-7 C, QSOX1S-1, QSOX1S-2 and (<b>F</b>) MDA-MB-231 shC, shQSOX1-1, shQSOX1-2 cells were transfected with the pGFP-LC3 vector. 24 h after transfection, cells were incubated with or without 100 nM bafilomycin A1 for 8 h. GFP-LC3 puncta were then analyzed by confocal microscopy. Each picture is representative of a typical cell staining observed in 10 fields chosen at random. Scale bar represents 20 µm. (<b>G and H</b>) GFP-LC3 puncta, observed in E and F respectively, were counted using the ImageJ software. For each group, 20 cells were randomly selected. Data are means ± S.D. of three independent experiments. *P<0.05 compared to the control.</p

Immunogenicity Evaluation of a Rationally Designed Polytope Construct Encoding HLA-A*0201 Restricted Epitopes Derived from <i>Leishmania major</i> Related Proteins in HLA-A2/DR1 Transgenic Mice: Steps toward Polytope Vaccine

<div><p>Background</p><p>There are several reports demonstrating the role of CD8 T cells against <i>Leishmania</i> species. Therefore peptide vaccine might represent an effective approach to control the infection. We developed a rational polytope-DNA construct encoding immunogenic HLA-A2 restricted peptides and validated the processing and presentation of encoded epitopes in a preclinical mouse model humanized for the MHC-class-I and II.</p><p>Methods and Findings</p><p>HLA-A*0201 restricted epitopes from LPG-3, <i>Lm</i>STI-1, CPB and CPC along with H-2Kd restricted peptides, were lined-up together as a polytope string in a DNA construct. Polytope string was rationally designed by harnessing advantages of ubiquitin, spacers and HLA-DR restricted Th1 epitope. Endotoxin free pcDNA plasmid expressing the polytope was inoculated into humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice intramuscularly 4 days after Cardiotoxin priming followed by 2 boosters at one week interval. Mice were sacrificed 10 days after the last booster, and splenocytes were subjected to <i>ex-vivo</i> and <i>in-vitro</i> evaluation of specific IFN-γ production and <i>in-vitro</i> cytotoxicity against individual peptides by ELISpot and standard chromium-51(⁵¹Cr) release assay respectively. 4 H-2Kd and 5 HLA-A*0201 restricted peptides were able to induce specific CD8 T cell responses in BALB/C and HLA-A2/DR1 mice respectively. IFN-γ and cytolytic activity together discriminated LPG-3-P1 as dominant, <i>Lm</i>STI-1-P3 and <i>Lm</i>STI-1-P6 as subdominant with both cytolytic activity and IFN-γ production, <i>Lm</i>STI-1-P4 and LPG-3-P5 as subdominant with only IFN-γ production potential.</p><p>Conclusions</p><p>Here we described a new DNA-polytope construct for <i>Leishmania</i> vaccination encompassing immunogenic HLA-A2 restricted peptides. Immunogenicity evaluation in HLA-transgenic model confirmed CD8 T cell induction with expected affinities and avidities showing almost efficient processing and presentation of the peptides in relevant preclinical model. Further evaluation will determine the efficacy of this polytope construct protecting against infectious challenge of <i>Leishmania</i>. Fortunately HLA transgenic mice are promising preclinical models helping to speed up immunogenicity analysis in a human related mouse model.</p></div

Percent of specific lysis of targets loaded by P1, P3 and P6 by T cell clones from individual mice at 30∶1 E/T effector to target ratio.

a<p>Responder to tested mice.</p>b<p>Specific lysis at 30∶1 effector to target ratio.</p><p>Percent of specific lysis of targets loaded by P1, P3 and P6 by T cell clones from individual mice at 30∶1 E/T effector to target ratio.</p

<i>In-vitro</i> evaluation of polytope expression using COS-7 cells.

<p>Recombinant pEFGP-PT was transiently transfected into COS-7 cells by means of linear Polyethylenimine 25 KDa. A1 represents COS-7 cells without any transfection, A2 represents COS-7 cells transfected with pEGFP-N3 as positive control and A3 represents COS-7 cells transfected with pEGFP-PT. Panel B represents the corresponding microscopic feature of each condition, B1 represents positive control and B2 represents COS-7 cells transfected with pEGFP-PT after 24 hours.</p

Balb/c response against 4 H-2Kd restricted peptides.

<p>4 mice were immunized with polytope construct three times with one week interval and sacrificed 10 days after the last booster. Splenocytes from individual mice were <i>in-vitro</i> re-stimulated by representative peptides (Kd1-4) of Balb/c and specific IFN-γ production was evaluated by <i>ex-vivo</i> ELISPOT assay. A. Representative of two experiments. Columns, mean of spots from duplicate wells for each mice from one representative experiment; bars, SD. Stimulations resulting in spots two times the negative control (unstimulated cells) and more than 10 were considered positive (stars). B. Statistical analysis of consolidated data from 4 mice against each individual peptide. The response against all 4 peptides appeared statistically significant compared to un-stimulated control cells (<i>p</i><0.05) with an exception for Kd4 which was subdominant in comparison to the rest. Horizontal lines represent the mean value. SFC: Spot Forming Cells.</p