Human neutrophils do not require transcription or translation to release NETs in response to <i>C</i>.<i>albicans</i>.

<p>(A, B) Inhibitors were used at the following concentrations: Actinomycin D (1 μg/ ml), flavopiridol (0.05 μM), CAS 577784-91-9 (10 μM), CHX (1 μg/ ml). Cells were infected with opsonized <i>C</i>.<i>albicans</i> at MOI 5 and subsequently fixed for immunofluorescence staining. (A) NET formation was quantified at 2h after infection by immunofluorescence staining of chromatin and Hoechst. Graphs show mean ± SEM from independent experiments with 3 different donors. (B) Representative images of an early time point (2h) of human neutrophils infected with <i>C</i>.<i>albicans</i> and treated with transcription or translation inhibitors. Cells were stained with PL2-3 (chromatin, red), NE (Neutrophil elastase, green) and Hoechst (blue). (C) Representative images of a late time point (4h) of <i>C</i>.<i>albicans</i>-infected neutrophils showing spread NETs that are unaffected by inhibitors of gene expression. (A) Statistical analysis revealed no significant changes after inhibitor treatment.</p

Human neutrophils do not require transcription or translation to release NETs in response to PMA.

<p>(A-G). Inhibitors were used at the following concentrations: Actinomycin D (1 μg/ ml), flavopiridol (0.05 μM), CAS 577784-91-9 (10 μM), CHX (1 μg/ ml). (A) Actinomycin D, flavopiridol and CHX, but not CAS 577784-91-9 inhibit <i>de novo</i> production of Mip-1α induced by LPS. (B) Actinomycin D and flavopiridol, but not CAS 577784-91-9or CHX block mRNA transcription of PMA-treated human neutrophils. (C.) Actinomycin D, flavopiridol and CHX, but not CAS 577784-91-9 inhibit <i>de novo</i> production of IL-8 induced by PMA. (D-G) Actinomycin D, flavopiridol, CAS 577784-91-9 or CHX do not block NET formation in response to PMA (100 nM). (D, E). NET formation was quantified by adding the DNA dye SYTO green (total cells) and the cell impermeable DNA dye SYTOX orange (NETs). (F) NET formation was quantified by immunofluorescence staining of chromatin and Hoechst as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157454#pone.0157454.ref010" target="_blank">10</a>]. Graphs show mean ± SEM from independent experiments with at least 3 different donors. (G) Representative images of human neutrophils stained with PL2-3 (chromatin, red), NE (Neutrophil Elastase, green) and Hoechst (blue) after treatment with transcription or translation inhibitors and PMA induction. (D-F) Statistical analysis revealed no significant changes after inhibitor treatment.</p

Mouse neutrophils do not require transcription or translation to release NETs.

<p>Inhibitors were used at the following concentrations: Flavopiridol (0.05 μM), CAS 577784-91-9 (10 μM), CHX (1 μg/ ml). (A) Flavopiridol and CHX, but not CAS 577784-91-9 block Mip-1α production in response to LPS. (B) PMA-induced NET formation is not inhibited by flavopiridol, CAS 577784-91-9 or CHX. Neutrophils isolated from Nox2 -/-mice are used as negative control. (C) NET formation in response to heat-killed <i>C</i>. <i>albicans</i> is not blocked by inhibitors. Graphs show mean ± SEM of experiments with neutrophils isolated from 3 mice. (D) Representative immunofluorescence pictures of murine neutrophils stained with PL2-3 (chromatin, red) and DAPI (blue) after infection with <i>C</i>. <i>albicans</i> (MOI 3) and treatment with transcription/ translation inhibitors. (B, C) Statistical analysis revealed no significant changes after inhibitor treatment.</p

The <i>var2csa</i> uORF is a repressive element.

<p>A. Constructs used in transient transfections. Constructs drive expression of either the firefly <i>luciferase</i> reporter gene, <i>Renilla luciferase</i> (in the case of control plasmid HRH) or both (in the case of V2RLH). VLH contains the un-modified promoter and upstream regulatory region from <i>var7b</i>. V2LH contains the un-modified promoter and upstream regulatory region from <i>var2csa</i>. V2LHm is identical to V2LH, except a single base pair mutation has altered the start codon of the upstream open reading (uORF) from ATG to ACG. In V2BLH, the uORF has been replaced with the <i>bsd</i> coding region while in V2RLH the uORF has been replaced by the coding region for <i>Renilla luciferase</i>. In V2ΔLH the entire region upstream of the uORF, including the transcription start site, has been deleted. In SURF the uORF has been shortened to 48 bp by the introduction of a premature stop codon, while in uORFL it has been lengthened to 450 bp by eliminating the endogenous stop codon. ICL contains the intact upstream regulatory region, including the uORF, however the intercistronic region has been duplicated. B. Levels of firefly luciferase expression from each construct shown in A. C. Levels of <i>Renilla</i> luciferase expression. V2RLH supports translation of robust levels of <i>Renilla</i> luciferase indicating that the uORF can be translated. The plasmid HRH, containing the strong <i>hrp3</i> promoter, was employed as a positive control for <i>Renilla</i> luciferase expression, while V2LH, which does not contain the <i>Renilla</i> luciferase gene, was a negative control. All assays were done simultaneously in triplicate.</p

Evidence for translational regulation of <i>var2csa</i> in cultured, wildtype parasites.

<p>A. The parasite population NF54 VAR2CSA was selected using anti-VAR2CSA rabbit antibodies. Flow analysis indicates that a majority of the population displays VAR2CSA on the cell surface (red) with a MFI value of 14.2 compared to the background (black) with a MFI value of 5.2 (arbitrary units). Flow analysis of NF54-239 indicates that the majority of the population displays low levels of VAR2CSA on the cell surface with a MFI value of 5.0 compared to the background value of 3.6 (arbitrary units). B. Selection for VAR2CSA expression results in recognition by antibodies predominantly from female sera whereas the NF54-239 line is poorly recognized. C. <i>var</i> transcript analysis shows that <i>var2csa</i> is the dominant transcript in both the NF54 VAR2CSA (Black) and Nf54-239 (white) parasite lines.</p

Growth rates of parasites translating different ORFs.

<p>Constructs are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000256#ppat-1000256-g004" target="_blank">Figure 4</a>. A. Parasites that have been selected for translation of the second open reading frame encoding <i>blasticidin-S-deaminase</i> (V2B+blasticidin) grow at the same rate as parasites not under blasticidin selection (V2B−blasticidin) or those in which translation of the uORF has been disrupted (V2mB+blasticidin). Parasites in which the uORF has been replaced with the coding region of <i>Renilla luciferase</i> (V2RB+blasticidin) grow at a slower rate. B. Parasites actively translating the second ORF encoding <i>blasticidin-S-deaminase</i> continue to display resistance to the drug if grown without selection pressure very briefly. C. When parasites are grown in the absence of blasticidin in the media for 21 days, they rapidly begin to repress translation of the second ORF and revert to the drug sensitive phenotype as demonstrated by slower growth when placed back under drug pressure (V2B+blasticidin). The histogram at the right shows that reversion to drug sensitivity is not due to a switch in promoter activity. Parasites grown without blasticidin pressure (V2B−BSD) for three weeks continue to express <i>bsd</i> mRNA at levels equal to or greater than those grown continuously under blasticidin pressure (V2B+BSD). <i>bsd</i> mRNA levels are shown as copy number normalized to <i>seryl-tRNA synthetase</i>.</p

Translational repression in stably transformed parasites.

<p>Parasites were stably transfected with either pVLH or pV2BLH (A). V2BLH contains the <i>bsd</i> selectable marker and VLH contains <i>hdhfr</i> for maintaining the episomes in transfected parasites (not shown). Integration into the genome occurred spontaneously and was selected for by alternate growth with or without drug. <i>luciferase</i> mRNA levels were determined using Q-RT-PCR (B) with three different primer pairs (1–3), whose corresponding location on the <i>luciferase</i> ORF is shown in (A). Levels of protein expression were assayed by measuring luciferase activity (C).</p

Selection for reversal of translational repression by the uORF of <i>var2csa</i>.

<p>A. Constructs used for selection of parasites that translate the second open reading frame of the <i>var2csa</i> gene. The drug selectable marker <i>blasticidin-S-deaminase</i> (BSD) was used to select parasites translating the downstream cistron (equivalent to exon I of <i>var2csa</i>). All three constructs also contain the <i>hdhfr</i> selectable marker for maintaining the episomes in transfected parasites prior to blasticidin selection (not shown). B. A drug resistant population appeared after six days suggesting that a subset of parasites is capable of translating the second ORF. Replacing the uORF with <i>Renilla luciferase</i> (V2RB) led to severely retarded growth in presence of blasticidin. Analysis of episomes recovered from these parasites indicated that they had undergone recombination (not shown).</p