Fig 2: Silencing of NOD1 or SSH1 results in diminished SOCS2 mRNA expression. (a/b) iDCs transfected with a control siRNA or with siRNA directed against (a) NOD1 or (b) SSH1 were stimulated with IL-10 (30 ng/ml) for 2 hours, and socs1, socs2 and socs3 mRNA expression was analysed by q-RT-PCR. Data represent mean and SD of five independent experiments. For statistical analysis a one-way ANOVA with Tukey’s post hoc test was performed.

Fig 3: Depletion of SSH1 is correlated with diminished STAT1 activation. (a/b) Immature DCs were generated and transfected with a control siRNA or siRNA directed against SSH1. 72 hours post transfection, cells were stimulated with IL-10 (30 ng/ml) for the indicated time points and STAT activation was analysed by Western Blot. Tyrosine phosphorylation of STAT1 and STAT3 was detected by specific antibodies. To control for equal loading, total STAT protein was detected. One out of four independent experiments (a) and quantification of four experiments (b) are shown. Data represent mean and SD. For statistical analysis a one-way ANOVA with Tukey’s post hoc test was performed.

Fig 4: Paladin interacts with slingshot homologue-1 and regulates the phosphorylation level of actin remodelling regulators in HCT116. A Paladin and serine 3 phospho-cofilin western blot in HCT116 shRNA clones. B mTOR and serine 2481 phospho-mTOR western blot in HCT116 shRNA clones. C Akt and serine 473 phospho-AKT western blot in HCT116 shRNA clones. D SSH1 western blot in HCT116 shRNA clones. HSC70 was used as a loading control. E Actin staining with fluorescent phalloidin in HCT116 shRNA clones. White arrows show filopodia. F, G HCT116 lysates were immunoprecipitated with SSH1, paladin or irrelevant antibodies (IP) followed by western blot with paladin, SSH1 or phospho-cofilin antibodies (WB).

Fig 5: Slingshot homolog-1 (SSH1)-mediated p62 inhibition at pSer403 increases small EV secretion. (A) Representative images of DiI-stained small EVs secreted from NIH3T3 cells transfected with control siRNA or SSH1 siRNA. (B) Quantification of secreted small EVs for (A). Data are presented as means ± SEM. n = 3 independent experiments, t-test, ***P = 0.0005. (C) Representative immunoblots for SSH1 and β-Actin protein levels in cell lysates for (A). (D) Representative images of DiI-stained small EVs secreted from NIH3T3 cells expressing VC or Flag-SSH1 (E) Quantification of secreted small EVs for (D). Data are presented as means ± SEM. n = 3 independent experiments, t-test, #P < 0.0001 (F) Representative immunoblots for Flag and β-Actin protein levels in cell lysates for (D). (G) Representative images of DiI-stained small EVs secreted from NIH3T3 cells co-transfected with VC or Flag-SSH1 and control siRNA or p62 siRNA. (H) Quantification of secreted small EVs for (G). Data are presented as means ± SEM. n = 3 independent experiments, 2-way ANOVA, followed by Sidak’s post-hoc, ***P = 0.0004. n.s. = not significant. (I) Representative immunoblots showing SSH1, p62, and β-Actin protein levels in cell lysates for (G). (J) Representative images of DiI-stained small EVs secreted from NIH3T3 cells co-transfected with VC or Flag-SSH1 and GFP, GFP-p62, or GFP-p62 S403E. (K) Quantification of secreted small EVs for (J). Data are presented as means ± SEM. n = 3 independent experiments, 2-way ANOVA, followed by Sidak’s post-hoc, ***P = 0.0007, **P = 0.001. n.s. = not significant. (L) Representative immunoblots for SSH1, p62, and β-Actin protein levels in cell lysates for (J).