Fig 2: EhCP5 signaling results in activation of MARCKS. A. LS174T cells were infected with live WTEh or EhCP5- for 15 minutes and subsequently immunostained for phospho-MARCKS and actin. Arrows indicate intracellular phospho-MARCKS in cells that are contacted by live WTEh. The dotted white line indicates the position of Eh. Arrows indicate key regions of the cell that contains activated intracellular MARCKS that is phosphorylated. Scale bar 10 μm. B. Quantification of confocal images was performed by measuring the concentration of threshold phosphorylated PKCδ staining within the host cell in the immediate contact site by Eh. C. Mucin granules were extracted and purified from LS174T cells previously stimulated with 50μg/mL-secreted components from WTEh along with subcellular fractionation of both the plasma membrane and cytoplasm. The following markers were used: Mucin for granule contents, Vamp8 for granule membrane, Integrin αv for the plasma membrane and GADPH for the cytoplasm. D. The gating strategy and calibration controls for flow cytometry of mucin granules using markers of known sizes 300nm (Blue), 1μm (Red) and presence of mucin within the granule as visualized with the lectin WGA (Black). E. Mucin granules were extracted from monolayers stimulated with 50μg/mL-secreted components from WTEh, EhCP5-, WTEh+E64 or PMA (1μM) for 20 minutes and analyzed by flow cytometry using the gating strategy shown in 5D. Mean fluorescence intensity was measured from PE-Phospho-MARCKS whereby PMA served as the positive control. F. siRNA was used to disrupt MARCKS and PKCδ protein expression at concentrations of 20, 5, 1nM and compared to 20nM non-targeting control pool (CP). GAPDH serves as a loading control. G. 3H-glucoasmine metabolically labeled LS174T cells were transfected with siRNA against MARCKS, PKCδ or control pool and assayed for mucin secretion in response to live Eh. Control cells were treated with the transfection reagent in the absence of siRNA. *** p <0.001, *p <0.05.

Fig 3: Loss of MARCKS in fibroblasts reduces its supporting of ovarian xenograft tumor growthA. Stably transfected MRC5-CAF cells were examined under phase-contrast microscopy (left panel) and fluorescent microscopy (right panel) to verify the >90% efficiency of lentiviral transfection. Western blot analysis confirmed the effect of MARCKS targeting lentivirus in repressing MARCKS expression. B. Representative bioluminescence images of mice (n=8 each group) bearing SKOV3-Luc cells alone, or co-injection with MRC5-CAFs stably transfected with either sh-NC or sh-MARCKS lentivirus at 4 weeks after tumor implantation. Bar graph showing the quantification of normalized total photon counts of the subcutaneous xenografts in mice of each group. C. H&E and D. Masson trichrome staining and E. Immunohistochemical staining of p-MARCKS, p-AKT, Twist1 and aSMA in tumor sections from mice in SKOV3-Luc cells solitary group, and in groups coinjected with sh-NC or sh-MARCKS lentivirus transduced MRC5-CAFs. F. A graphical illustration of the molecular signaling events involved in MARCKS regulation of CAF activation. MARCKS overexpression suppresses cellular senescence and boosts the activation of AKT/Twist1 signaling to maintain the CAF features, thus supporting tumor cells growth and invasion. (*P<0.05; **P<0.01; ***P<0.001).

Fig 4: Inhibition of MARCKS attenuates the supportive role of CAFs to tumor cellsA. Representative images and statistical analysis of cellular migration of SKOV3 and primary ovarian cancer cells, in coculture invasion system with MRC5-CAFs transfected with si-NC or si-MARCKS. B. Representative fluorescent images and statistical analysis of the relative cell numbers of PKH-26 (red) labeled SKOV3 and primary ovarian cancer cells, after 72 h contact coculture on top of the CMAC (blue) labeled MRC5-CAFs transfected with si-NC or si-MARCKS. C. Schematic depicting of the 3D organotypic culture assay with matrigel ECM substrate, fluorescently labeled CAFs (red, PKH-26), HMrSV5 mesothelial cells (blue, CMAC) and tumor cells (green, PKH-67) from bottom to the top layer. D. Representative fluorescent images and quantification of the proliferation index of PKH-67 labeled SKOV3 and primary ovarian cancer cells, in co-culture model as described above with variant MRC5-CAFs transfected with si-NC or si-MARCKS. Data are expressed as mean ± s.e.m. of at least three independent experiments. (**P<0.01; ***P<0.001).

Fig 5: Ectopic expression of wild‐type (WT) MARCKS, but not the pseudophosphorylation mutant rescues the migration defects observed in MARCKS KD in dedifferentiated VSMCs. A, Dedifferentiated rat A10 VSMCs were treated with control siRNA (Ctl) or MARCKS siRNA (KD) and cotransfected with plasmids for either pEGFP‐N1 vector alone (Vec), WT bovine MARCKS (MARCKS WT), the myristoylation‐deficient MARCKS mutant (G2A), the phosphorylation‐deficient ED domain MARCKS mutant (S4G), or the pseudophosphorylated ED domain MARCKS mutant (S4D), respectively. B, Cell migration was determined by the wound‐healing assay in normal growth media as described in the Materials and Methods section. Cotransfection with WT MARCKS, myristolation‐deficient MARCKS, and phosphorylation‐deficient MARCKS all rescued migration. The pseudophosphorylation mutant did not rescue migration. Data are presented as the mean and SE of 3 independent experiments and normalized to the control treatment (Ctl+Vec). *P<0.05. C, Activation of the GTPases Rac1 and Cdc42 was determined in the presence of MARCKS KD and cotransfection with WT plasmids and each of the 3 mutant plasmids. D, The WT MARCKS, G2A, and S4G, but not the pseudophosphorylated S4D mutant, rescued both Rac1 and (E) cdc42 activation. Data are presented as the mean and SE of 3 independent experiments normalized to the quiescent condition (−PDGF) in control cells (Ctl+Vec). *P<0.05. F, PDGF stimulates MARCKS phosphorylation (Pi‐MARCKS) in CASMCs in a time‐dependent manner. Maximal MARCKS protein expression was observed after 10 minutes of stimulation. G, MARCKS phosphorylation is also dependent on the dose of PDGF. Exposure of starved cells to 50 ng/mL yielded the greatest phosphorylation of MARCKS at 10 minutes (right panel). CaM indicates calmodulin; ED, effector domain; GFP, green fluorescent protein; KD, knockdown; MARCKS, myristoylated alanine‐rich protein kinase substrate; PDGF, platelet‐derived growth factor; pEGFP, enhanced green fluorescent protein plasmid; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell; WT, wild type.