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

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

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

“Race for the Surface”: Eukaryotic Cells Can Win

With an aging population and the consequent increasing use of medical implants, managing the possible infections arising from implant surgery remains a global challenge. Here, we demonstrate for the first time that a precise nanotopology provides an effective intervention in bacterial cocolonization enabling the proliferation of eukaryotic cells on a substratum surface, preinfected by both live Gram-negative, <i>Pseudomonas aeruginosa</i>, and Gram-positive, <i>Staphylococcus aureus</i>, pathogenic bacteria. The topology of the model black silicon (bSi) substratum not only favors the proliferation of eukaryotic cells but is biocompatible, not triggering an inflammatory response in the host. The attachment behavior and development of filopodia when COS-7 fibroblast cells are placed in contact with the bSi surface are demonstrated in the dynamic study, which is based on the use of real-time sequential confocal imaging. Bactericidal nanotopology may enhance the prospect for further development of inherently responsive antibacterial nanomaterials for bionic applications such as prosthetics and implants