Anti-HIV-1 activity and cytokine release by ProTα variants are not entirely TLR4-dependent.

<p>(A) Murine or (B) human macrophages untreated or treated with the specific TLR4 inhibitor small molecule TAK-242 (3 μM) for 2 h, were stimulated with 20 ng of ProTα variants for 2 h and then infected with HIV-1_VSV (MOI: 0.01) expressing the Luciferase reporter gene_. Anti-HIV-1 activity of ProTα variants was assessed on cell lysates 48 h post-infection by luciferase assay normalized for the protein concentration. Supernatants from infected human macrophages treated with ProTα variants in the presence or absence of TAK-242 were collected 6 hours and 48 hours post-infection and used to measure the release of (C) IFN-β, and (D) IL-6 and (E) TNF-α, respectively. Each experiment was repeated at least three times. Data represent mean ± SD. Student’s <i>t</i> test was used to compare means of treated versus control samples. *<i>P</i><0.05 was considered to be statistically significant.</p

Up-regulation of mRNA of different innate inhibitors of viral replication in primary human macrophages treated with ProTα variants.

<p>A) INF-β and IFN-λ1 and (B) TNF-α, IL-6 and RANTES mRNAs induction by ProTα variants. RT-qPCR with specific primers for indicated genes was performed as detailed in Methods on cDNA from primary human macrophages treated for 2 h with 200 ng/ml of ProTα variants or LPS. mRNA transcripts levels were normalized relatively to the expression of the ribosomal protein (RPS11) control mRNA. Each experiment was done at least three times. Data represent mean ± SD. Student’s <i>t</i> test was used to compare means of treated versus control samples. *P < 0.05 was considered to be statistically significant.</p

Silver stained PAGE of recombinant ProTα variants and amino acid sequence alignment of ProTα variants.

<p>Left panel: silver staining protein visualization of PAGE in which 0.5 μg of each recombinant ProTα variant was loaded. Right panel: alignment of amino acid sequence of ProTα variants. The peptide sequences identified by mass spectrometry from silver stained gel bands are in grey.</p

Mass spectrometry identification of the amino acid sequences and frequency of peptides (in brackets) released by PBMCs in response to HIV infection.

<p>Mass spectrometry identification of the amino acid sequences and frequency of peptides (in brackets) released by PBMCs in response to HIV infection.</p

TLR4-dependent stimulation of IFN-β by ProTα variants.

<p>(A) IFN-β, (B) IL-6, and (C) TNF-α expression was measured by RT-qPCR on cDNAs from wild type and TLR4^-/- mouse bone marrow-derived macrophages treated with 200 ng/ml of different ProTα variants for 2 h. Though at lower levels, TNF-α remains inducible by the variants in TLR^-/- murine macrophages, suggesting the possible involvement of other receptor(s).</p

ProTα variants are rapidly released in response to HIV infection and PolyI:C treatment.

<p>Release of ProTα variants by ELISA (A and B) and control MTS cytotoxicity assay (C) were performed on 24 h supernatants from mock or HIV-1_BaL-infected (MOI 0.01) primary human CD4⁺ T cells and ectocervical cells (Ecto). Cells treated for 24 h with the DNA-damaging agent etoposide (2 μM) were used as a positive control in the cytotoxicity assay (C). (D) Mass spectrometry analysis of the variants’ specific peptides present in the supernatants from healthy donor-derived PBMCs uninfected or 10, 20, 30, 60 min post-infection with HIV-1_BaL at MOI 0.01 showed the rapid release of the ProTα variants.</p

ProTα variants are predicted to be intrinsically disordered proteins with high binding promiscuity.

<p>(<b>A</b>) Per-residue disorder distribution in variants of human ProTα (green line and shade–iso2; yellow line and shade–isoB variant; red line and shade–p7 ProTα variant). Disorder propensity was evaluated by PONDR-FIT. Scores above 0.5 correspond to disordered residues/regions. Profiles were aligned to match some specific disorder-based feature. Shades represent distribution of errors in disorder estimation by PONDR-FIT. (<b>B</b>) Evaluation of the experimental and predicted interaction of the human iso2 (UniProt ID: P06454) by STRING (Search Tool for the Retrieval of Interacting Genes) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156486#pone.0156486.ref028" target="_blank">28</a>], which produces the network of predicted associations for a particular group of proteins. The network nodes are proteins and the edges represent the predicted functional associations. An edge may be drawn with up to 7 differently colored lines each representing one of the seven types of evidence used in predicting the associations. A red line indicates the presence of fusion evidence; a green line—neighborhood evidence; a blue line–co-occurrence evidence; a purple line—experimental evidence; a yellow line–text mining evidence; a light blue line—database evidence; a black line–co-expression evidence [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156486#pone.0156486.ref028" target="_blank">28</a>].</p

Evaluation of functional disorder in human ProTα and its variants analyzed in this study.

<p>Evaluation of functional disorder in human ProTα and its variants analyzed in this study.</p

LPS exposure increases IL6 expression and STAT3 activation in ATG7KO RTEC cells.

<p>qPCR analysis demonstrated increased LPS-induced IL-6 expression in cortex of ATG7KO kidneys compared to LPS injected controls (A) which was accompanied by (B) decreased LC3-II and increased STAT3 phosphorylation. (C) Densitometric analysis of the expression of phospho-STAT3 relative to total STAT3. (D)Western blot of FKT- and FKT+ cells indicated that the ablation of Atg7 impaired accumulation of the LC3-II isoform in response to LPS (D). At the same time, qPCR analyses showed that the LPS induction of IL-6 was enhanced in the ATG7 deficient FKT- cell line (E) and in RTEC isolated from wild type mice co-incubated in the absence or presence of 5mM 3MA (F).</p

Association between CB₁R and DOR alters CB₁R localization.

<p><b>A</b>, Lysates (100–200 µg) from N2A^CB1R and N2A^CB1RDOR cells were subjected to immunoprecipitation with 1 µg of anti-CB₁R (C-terminal) antibody, the immunoprecipitates were resolved on 10% SDS-PAGE and probed for the presence of myc-DOR using mouse monoclonal anti-myc antibody (1∶1000) and for CB₁R using rabbit polyclonal anti-CB₁R (C-terminal) antibody (1∶500) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). Representative of 3 independent experiments shown. <b>B</b>, CB₁R-DOR complexes exhibit greater interaction with AP-2 than AP-3. Lysates (100–200 µg) from N2A^CB1R and N2A^CB1RDOR cells were subjected to immunoprecipitation using 1 µg of an anti-CB₁R (C-terminal) antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. The immunoprecipitates were resolved on 10% SDS-PAGE and probed for the presence of AP-3 (1∶1000), AP-2 (1∶1000) and CB₁R (C-term) (1∶500) using specific antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). Representative of 3 independent experiments shown. <b>C</b>, Localization of endogenous CB₁R in N2A^CB1R and of CB₁R and DOR in N2A^CB1RDOR cells. Cells fixed with 4%PFA in PBS and permeablized with 0.1% Triton, were stained with the rabbit polyclonal anti-CB₁R (C-terminal) antibody (1∶500; green) and the mouse monoclonal anti-myc antibody (1∶1000; red) and visualized using Alexa 488-coupled anti-rabbit or Alexa 594-coupled anti-mouse secondary antibodies (1∶1000) using confocal microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. Representative of 3 independent experiments shown. <b>D</b>, Cell surface staining of endogenous CB₁R and stably expressed DOR in N2A^CB1RDOR cells. N2A^CB1R and N2A^CB1RDOR cells were stained with a goat polyclonal anti-CB1R (N-terminal) antibody (1∶500) and mouse monoclonal anti-myc antibodies (1∶1000) prior to fixation of the cells to label cell surface receptors, as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#pone.0029239-Rozenfeld1" target="_blank">[28]</a>. After fixation, cells were visualized with Alexa 594-coupled anti-goat and Alexa 488-coupled anti-mouse secondary antibodies (1∶1,000) using confocal microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. Representative of 3 independent experiments shown.</p