Figure 1.
HTS siRNA-screening identifies SSH1 as novel component of the NOD1-pathway.
(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 Figures S1, S2 and Table S1).
Figure 2.
SSH1 is essential and specific for NOD1-mediated signaling.
(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 Figure S4).
Figure 3.
SSH1 is involved in early Shigella-induced pro-inflammatory responses.
(A) HeLa cells were transfected with CTRL or SSH1 siRNAs and incubated for 72 h and infected with Shigella flexneri 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 S. flexneri 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 Figure S5).
Figure 4.
SSH1 interacts with NOD1 and regulates cofilin phosphorylation after NOD1 activation.
(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) In situ 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 S. flexneri (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 Figure S6).
Figure 5.
ROCK activity influences NOD1 signaling.
(A) HEK293T cells were transfected with CTRL or siRNA specific for the indicated target and incubated for 48 h. Subsequently, cells were transfected with the NF-κB luciferase reporter and NOD1 and stimulated with TriDAP (filled bars). After 16 h, cells were lysed, luciferase activation was determined and normalized with the β-galactosidase values (nRLU). Values are mean + S.D. (n = 3). Knock-down of the respective target is shown by immunoblot in the right panel. (B) Effect of the expression of constitutively active (CA) and dominant negative (DN) forms of ROCK1 on NOD1-mediated NF-κB activity measured by luciferase reporter assays in HEK293T cells. Values are mean + S.D. (n = 3). (C) NOD1-induced NF-κB activity measured by luciferase reporter assays in HEK293T cells for 6 h with the ROCK inhibitors Y-27632 and Glycyl-H1152. Values are mean + S.D. (n = 3). (D) THP1 cells were treated with the indicated amount of ROCK inhibitor Glycyl-H1152 for 6 h and TriDAP-induced IL-8 secretion was determined by ELISA. Mean + S.D. (n = 3) is shown. Cells were lysed and p-cofilin was detected by Western Blot analysis using a specific antibody (lower panel). Detection of GAPDH served as loading control. (E) Same experimental setting as (D), using HeLa cells stimulated with 10 µg/ml TriDAP. Mean + S.D. is shown. * p<0.05, ** p>0.005 compared to Ctrl (see also Figure S8).
Figure 6.
NOD1 signaling is dependent on F-actin.
(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.