Fig 2: 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 3: SSH1 inhibition suppresses the viability, oncogenicity, and cancer stemness of HCC cells. (A) Representative western blot images and histograms comparing cell viability in THLE-2, Huh7-WT, Huh7-SSH1-/-(1), and Huh7-SSH1-/-(2) cells at 100x and 200x magnification. (B) Molecular structure of Sennoside A with a molecular weight of 862.74 g/dl. (C) Histograms of the cell viability of Huh7, HepG2, Mahlavu, or Hep3B cells treated with 0 - 100 μM Sennoside A for 24h or 48h, with IC50 indicated. (D) Representative wound-healing migration images and histograms comparing the wound-gap closure in Huh7, Huh7-SSH1-/-(1), Huh7-SSH1-/-(2), 20 μM or 40 μM SenA-treated Huh7 cells. (E) Representative invasion assay images and histograms comparing the number of invaded Huh7, Huh7-SSH1-/-(1), Huh7-SSH1-/-(2), 20 μM or 40 μM SenA-treated Huh7 cells. (F) Representative colony-formation assay images and histograms comparing the number of colonies formed by Huh7, Huh7-SSH1-/-(1), Huh7-SSH1-/-(2), 20 μM or 40 μM SenA-treated Huh7 cells. (G) Representative tumorsphere-formation assay images and histograms comparing the number of tumorspheres formed by Huh7, Huh7-SSH1-/-(1), Huh7-SSH1-/-(2), 20 μM or 40 μM SenA-treated Huh7 cells. WT, wild type; SenA, Sennoside A; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: 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 5: Slingshot homolog-1 (SSH1) increases small EV secretion partially through cofilin activation and F-actin disruption. (A) Schematic of SSH1 full length, SSH1ΔC, and SSH1ΔN proteins. (B) Representative images of DiI-stained small EVs secreted from NIH3T3 cells expressing VC, Flag-SSH1, Flag-SSH1ΔN, or CFP-SSH1ΔC. (C) Quantification of secreted small EVs for (B). Data are presented as means ± SEM. n = 3 independent experiments, 1-way ANOVA, followed by Dunnett’s post-hoc, #P < 0.0001, ***P = 0.001, **P = 0.0069. (D) Representative immunoblots for Flag, GFP, and β-Actin protein levels in cell lysates for (B). (E) Representative images of DiI-stained small EVs secreted from NIH3T3 cells expressing GFP or GFP-Cofilin. (F) Quantification of secreted small EVs for (E). Data are presented as means ± SEM. n = 3 independent experiments, t-test, #P < 0.0001. (G) Representative immunoblots for cofilin and β-Actin protein levels in cell lysates for (E). (H) Representative images of DiI-stained small EVs secreted from NIH3T3 cells expressing GFP, GFP-cofilin-S3A, or GFP-cofilin-S3E. (I) Quantification of secreted small EVs for (H). Data are presented as means ± SEM. n = 3 independent experiments, 1-way ANOVA, followed by Dunnett’s post-hoc, #P < 0.0001. (J) Representative immunoblots for GFP and β-Actin protein levels in cell lysates for (H). (K) Representative images of DiI-stained small EVs secreted from NIH3T3 cells transfected with control siRNA or cofilin siRNA. (L) Quantification of secreted small EVs for (K). Data are presented as means ± SEM. n = 3 independent experiments, t-test, #P < 0.0001. (M) Representative immunoblots for cofilin and β-Actin protein levels in cell lysates for (K). (N) Representative images of DiI-stained small EVs secreted from NIH3T3 cells with or without 100 nM Latrunculin B (LatB) or 100 nM Jasplakinolide (Jasp) treatment for 8 h. (O) Quantification of secreted small EVs for (N). Data are presented as means ± SEM. n = 3 independent experiments, 1-way ANOVA, followed by Dunnett’s post-hoc, #P < 0.0001. n.s. = not significant.