Clone size of randomized shRNA-libraries.

<p>To indicate the complexity of the shRNA library, the number of bacterial colonies of each transformation step (primary library, secondary library and final library) was documented as colony forming units (cfu).</p

Specificity of shRNAs against HIV-1.

<p><b>a)</b> Designation of the identified shRNAs <b>b)</b> shRNA-target sequence of the HIV-1 proviral clone pNL4.3 <b>c)</b> starting nucleotide of the shRNA based on +1 being the transcriptional start site. <b>d)</b> Number of known isolates which include the target sequence.</p

Construction of the shRNA-library.

<p><b>a</b>) The 3 HIV segments of pNL4.3 were fragmented using DNaseI and blunt-ended. <b>b</b>) Fragments of 150–300 bp were eluted and ligated to the 3′ loop. To limit the size of the HIV-1 inverted repeats, a recognition site for MmeI, which cleaves exactly 20nt from its recognition site and leaves 2 nt 5′ overhangs, was engineered into the 3′ loop. <b>c</b>) Ligation of the 5′ loop to the MmeI-digested fragments generated a quasi-circular single-stranded structure. <b>d</b>) Rolling circle amplification (RCA) reactions using Φ 29 DNA polymerase and the primers RCA1 and RCA2 were performed to amplify the single-stranded circular DNA and to generate the complementary strand yielding a DNA concatemere of palindromic, inverted repeats encoding siRNA molecules. Digestion with BglII and MlyI liberated shRNA sequences which were inserted into the expression vector pENTR/siLib. <b>d</b>) The shRNA sequences were cloned into the linearized pENTR/Lib generating the primary library which was religated after BamHI digestion yielding the secondary library. The final lentiviral shRNA-library was generated by LR recombination of the secondary library into pL/EGP/siLib.</p

Reconfirmation of the inhibitory potential of selected shRNA-sequences.

<p><b>a</b>) ShRNAs were PCR-amplified from genomic DNA of selected cell clones as shRNA expression cassettes consisting of H1 promoter, shRNA-sequence and polyT. Each of the 200 individual cassettes were co-transfected with the HIV-1 specific Luciferase reporter construct pNL4.3Luc.R-E- into HEK 293 FT cells. Luciferase expression was measured 48 h p.t. Cells transfected with pNL4.3Luc.R-E- and a scrambled shRNA (sh scr) expressing cassette were used as control. <b>b</b>) Map of the identified shRNAs and re-evaluation of their inhibitory potential upon co-transfection as described in <b>a</b>). Cells which were transfected with pNL4.3LucR-E- alone or co-transfected with a scrambled siRNA control served as controls. Error bars indicate +/− SD of mean of three independent experiments.</p

Expression of the shRNAs from different polymerase III promoters.

<p><b>a</b>) 6 potent shRNA sequences were subcloned to allow for U6 promoter driven shRNA expression. U6 or H1 promoter expression vectors and pNL4.3LucR-E- were co-transfected into HEK 293 FT cells. Western blot analysis with a HIV-1 Integrase specific antibody 48 h p.t. indicated that the shRNAs can be efficiently expressed from both polymerase III promoters. Actin was used as loading control. <b>b</b>) <b>c</b>) Published shRNA-sequences against pol, nef, rev/env, gag and vpu/env were cloned to be expressed by the U6 promoter. These constructs as well as 14 library shRNAs and pNL4.3LucR-E- were co-transfected into HEK 293 FT cells and analysed 48 h post transfection. Western blot analysis <b>b</b>) with a HIV-1 Integrase specific antibody or <b>c</b>) luciferase assays demonstrated that the newly identified shRNAs are as potent as the published shRNAs. A scrambled shRNA (sh scr) and non-transfected cells were used as controls.</p

SSH1 interacts with NOD1 and regulates cofilin phosphorylation after NOD1 activation.

<p>(A) Co-immunoprecipitation of ectopically expressed SSH1 with NOD1 and NOD2 in human HEK293T cells. Flag-SSH1 was precipitated using a Flag-specific antibody in all samples. (B) Co-immunoprecipitation kinetic of NOD1 and SSH1 in HEK293T cells treated for the indicated time with 500 nM TriDAP. Flag-SSH1 was precipitated using a Flag-specific antibody. (C) <i>In situ</i> PLA detection of the interaction between GFP-SSH1 (shown in green) and Flag-NOD1 in transiently transfected HeLa cells. Protein–protein interactions are visualized as small, distinct red spots (PLA signals). F-Actin (blue) was stained with Alexa633-labeled phalloidin. Shown is a single z section. Scale bar 5 µm. Right panel: Quantification of the total spot area per cell. Significance was calculated by student's t-test (unpaired, two-tailed), p<0.0001. (D) Indirect immunofluorescence study of ectopically expressed myc-SSH1 in HeLa cells after 30 min of infection with <i>S. flexneri</i> (MOI = 10). Bacteria were stained with a LPS specific antibody and are shown in red in the merge image together with DNA staining in blue. Bar: 10 µm. (E) HEK293T cells expressing low amounts of NOD1 were stimulated with TriDAP. IL-8 release (bars) was measured by ELISA (accumulation in supernatant since t = 0) and IL-8 mRNA (line) by qPCR (upper panel). Values are mean + S.D. (n = 3). Cofilin phosphorylation (S3) was monitored by Western Blot analysis (lower panels). Probing for total cofilin and actin served as loading controls. One representative experiment out of three is shown. (F) p-cofilin and total cofilin protein levels were analyzed in HEK293T cells at the indicated time-points after stimulation with 50 ng/ml TNF (upper panel). Alternatively NOD1 was silenced by siRNA treatment for 48 h and cells were stimulated with TriDAP (lower panels) (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004351#ppat.1004351.s006" target="_blank">Figure S6</a>).</p

HTS siRNA-screening identifies SSH1 as novel component of the NOD1-pathway.

<p>(A) Combined Z-scores of the primary-hits in the counter screen with TriDAP (NOD1,ordinate) and TNF (abscissa) activation. (B) Flow-chart representing the screening procedure. Number of hits (genes) of each step is indicated. (C) Final hitlist ranked according to the THP1 results (best). Candidates validated in all steps and not affecting TNF signaling are shown in bold. Z-score were normalized to control siRNAs set to 0. (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004351#ppat.1004351.s001" target="_blank">Figures S1</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004351#ppat.1004351.s002" target="_blank">S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004351#ppat.1004351.s009" target="_blank">Table S1</a>).</p

SSH1 is involved in early <i>Shigella</i>-induced pro-inflammatory responses.

<p>(A) HeLa cells were transfected with CTRL or SSH1 siRNAs and incubated for 72 h and infected with <i>Shigella flexneri</i> M90T or BS176 (MOI = 10). IL-8 and IL-6 (right panel) secretion was determined 6 h post infection by ELISA. Values are mean + S.D. (n = 2), representative of three independent experiments. Knock-down efficiency was determined by RT-PCR (right panel). (B) Intracellular bacteria were counted after gentamycin treatment of the cells treated as in (A) 2 h post infection. (C) HeLa cells treated for 72 h with siCTRL (filled bars) or siSSH1_1 (gray bars) were analyzed at the indicated time-points after infection with <i>S. flexneri</i> M90T afaE. Quantitative PCR of cDNA normalized to GAPDH expression and IL-8 concentration in the supernatant (right panel), mean + S.D. (n = 3) are shown. Significance was calculated by student's t-test (unpaired, two-tailed) *p<0.05, **p<0.005 (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004351#ppat.1004351.s005" target="_blank">Figure S5</a>).</p

SSH1 is essential and specific for NOD1-mediated signaling.

<p>(A–B) PMA differentiated THP1-blue cells were treated for 72 h with a non-targeting (siCTRL) or two SSH1-specific siRNA duplex and incubated with TriDAP (10 µg/ml), MDP (10 µg/ml), TNF (0.1 µg/ml), or LPS (0.05 µg/ml). (A) NF-κB activation was measured by SEAP secretion. (B) IL-8 levels in the culture supernatants of the cells from (A). Values are mean + S.D. from two independent experiments conducted in triplicates. Significance was calculated by student's t-test (unpaired, two-tailed) *p<0.05, **p<0.005. n.s.: not significant. (C) Immunoblot of one of the experiments from (A), probing for SSH1 and actin as loading control is shown. (D) Early effects of the knock-down of SSH1. IL-8 (left panel) and SSH1 (right panel) mRNA levels in THP1-blue cells treated as inducted were measured by qPCR. Mean + S.D. from triplicate measurements of one representative experiment is shown (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004351#ppat.1004351.s004" target="_blank">Figure S4</a>).</p

NOD1 signaling is dependent on F-actin.

<p>(A) Effect of 6 h cytochalasin D (cytoD) treatment on TriDAP/NOD1 induced NF-κB activation in PMA differentiated THP1-blue reporter cells. Mean + S.D. (n = 3). Significance was calculated by student's t-test (unpaired, two-tailed) *p<0.05, **p<0.005 (compared to 0 nM cytoD). (B) IL-8 secretion of THP1 cells treated for 6 h with the indicated PAMP in the presence or absence of cytochalasin D. Shown is the mean of triplicates from two independent experiments + S.D. (n = 6). The fold induction of IL-8 by cytoD is depicted below. (C) SSH1 knock-down effect on cytoD enhanced NOD1 signaling. Differentiated THP1 cells were treated with cytoD for 6 h and stimulated with 10 µg/ml TriDAP (black bars) or mock treated (open bars). IL-8 secretion measured in the supernatant after 6 h of incubation is shown, mean + S.D. (n = 3), * p<0.05, ** p<0.005.</p