Specific nucleoporins are degraded in HRV16-infected cells.

<p>(<b>A</b>) Ohio-HeLa cells were infected without (mock) or with HRV16 (MOI of 1) and cells lysed using RIPA buffer containing protease and phosphatase inhibitors at the time points shown. Cell lysates were subjected to SDS-PAGE on 4–20% gradient gels and Western analysis using the indicated primary antibodies/horseradish peroxidise-conjugated secondary antibodies and enhanced chemiluminescence (Perkin Elmer). The specificity of the antibodies is indicated on the left. Bands corresponding to 3C, 3CD’ and 3CD are indicated on the right. p.i. - post-infection. (<b>B</b>) Results for densitometric analysis of FG-Nup protein bands (left) and non-FG-Nups (right) such as those shown in (A), where data were normalised to the corresponding values for tubulin, relative to the corresponding values for the mock sample. Densitometric analyses were performed using Image J; values represent the mean (± SD) from two independent experiments.</p

Active 3C protease is sufficient to degrade nuclear proteins/nucleoporins in transfected cells.

<p>(<b>Ai</b>) COS-7 cells transfected to express either GFP-3C or GFP-3Cinac were trypsinized 18 h after transfection, harvested in ice-cold PBS and FACS sorted to collect GFP-expressing cells. Cells were then lysed using RIPA buffer plus protease and phosphatase inhibitors and lysates were subjected to Western analysis as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g001" target="_blank">Figure 1A</a>; untransfected cells were lysed similarly and used as control. The antibodies used are shown on the left of the figure. The arrow in the nucleolin blot denotes a clear cleavage product, with the approximate molecular weight (kDa) indicated on the right. (<b>Aii</b>) Results for densitometric analysis of nucleolin and Nup153 protein bands such as those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g003" target="_blank">Figure 3A</a>i, where data were normalised to the corresponding value of tubulin, relative to the corresponding value for the control sample. Densitometric analyses were performed using ImageJ; values represent the mean (+ SD) from two independent experiments (<b>Bi</b>) Ohio-Hela whole cell lysates were incubated with 4 units of HRV14 3C protease at 37°C for the indicated incubation times, subsequent to SDS-PAGE on 10% gels and Western analysis as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g001" target="_blank">Figure 1A</a>. (<b>Bii</b>) Plot of densitometric analysis of protein bands in (Bi), where data were normalised to the corresponding values for tubulin, relative to 0 h samples. Densitometric analyses were performed using Image J and values were the mean of two different experiments (± SD). (<b>C</b>) COS-7 cells transfected to express either GFP-3C or GFP-3Cinac were fixed and permeabilized 18 h post-transfection, and immunostained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g002" target="_blank">Figure 2</a> with the indicated primary and Alexa-568 conjugated secondary antibodies. Fluorescence was imaged by CLSM (see Materials and Methods). In each panel, images on the left depict localisation of HRV16 proteins (green channel) and the images in the middle depict localisation of cellular proteins (red channel), with the merged image on the right.</p

HRV16 infection leads to mislocalisation of nuclear proteins.

<p>Ohio-HeLa cells grown on coverslips were infected without (mock) or with HRV16 as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g001" target="_blank">Figure 1</a>; cells were fixed at the indicated times and permeabilized, and then probed with the indicated pairs of primary antibodies, followed by Alexa 488 and Alexa-568 conjugated secondary antibodies. Fluorescence was imaged by CLSM (see Materials and Methods). In each panel, images on the left depict localisation of HRV16 proteins (green channel) and the images in the middle depict localisation of cellular proteins (red channel), with the merged image on the right.</p

Production of extracellular traps is associated with ROS production and is inhibited by DNase.

<p>Panels A shows that neutrophils expressing NETs have a four-fold increase in ROS fluorescence compared to NET negative (-ve) cells (NET-ve 16 706 (5879–42 332), NET+ve 64 146 (44 738–122921)), (n = 7, p = 0·008). Panel B shows that macrophages expressing MET-like structures have a two-fold increase in ROS fluorescence compared to MET negative (-ve) cells (MET-ve 45 644 (6257–118907), MET+ve 93 636 (28 113–392 973)), (n = 8, p = 0·023). Panels C and D demonstrate inhibition of NET (NTHi 44 (24–53), NTHi &amp; DNase 3 (0–10)), (n = 6, p = 0·004) and MET-like structure (NTHi 19±5, NTHi &amp; DNase 0.1±0.1)), (n = 8, p = 0·008) formation by the addition of DNase.</p

Baseline characteristics of the BAL patients.

<p>Baseline characteristics of the BAL patients.</p

Response by human bronchoalveolar macrophages to nontypeable <i>Haemophilus influenzae</i>.

<p>Panel A shows adherent BAL macrophages with staining for NTHi and production of ROS fluorescence in macrophages infected with NTHi by confocal microscopy. Panel B shows the ROS production in control (uninfected) macrophages and NTHi-infected macrophages over a 17 hour time-period (n = 15 subjects for each time-point) measured by confocal microscopy. At each one-hour time-point ROS production is significantly higher in the NTHi group (p&lt;0·01). Panel C shows the increase over 17 hours in ROS production (1 hour 13012±2214, 17 hours 15175±2247)), (n = 15, p = 0·009). Panel D shows upregulation of ROS as measured by the effect of three different strains of NTHi using chemiluminescence (control 5937 (1839–10855), NTHi-1 8193 (3569–15538), NTHi-2 8953 (5105–17111), NTHi-3 10093 (4136–20848)), (RLUs = reactive light units/second). The chemiluminescence predominantly measures extracellular ROS production. Panels E and F demonstrates an examples of the presence of high and low-producing ROS fluorescence populations in control (unstimulated) and NTHi-stimulated macrophages from a patient (using flow cytometry). The high-producing ROS populations have significantly increased surface expression of the M1 marker HLA-DR (p&lt;0·001): Panel G shows the unstimulated (baseline) macrophage population (low peak 6 (1–45), high peak 98 (94–99)), (n = 17), whilst Panel H shows the NTHi-stimulated macrophage population (low peak 11 (7–40), high peak 95 (80–100)), (n = 15).</p

Production of phagocyte extracellular traps and proteases in response to nontypeable <i>Haemophilus influenzae</i>.

<p>Panel A shows the % of neutrophils expressing neutrophil extracellular traps (NETs), (control 7 ±2, NTHi 40±6)), (n = 7, p = 0·002)) and Panel B shows the % of BAL macrophages expressing macrophage extracellular traps-like structures (METs), (control 0, NTHi 19±5), (n = 8, p = 0·008); after stimulation with NTHi compared to control. Panel C shows neutrophils producing NETs with co-expression of neutrophil elastase (arrow). Panel D shows macrophages producing a MET with co-expression of macrophage metalloproteinase-12 (arrow). Panel E shows that neutrophils (40±6, n = 7) have a higher expression of extracellular traps compared to macrophages (19±5, n = 8) (p = 0·013). Panel F; NTHi increases surface expression of MMP12 by BAL macrophages as measured by flow cytometry (control 804±78, NTHi 1075±142)), (n = 8, p = 0·005).</p