Fig 2: NCAM antibody and the MARCKS-derived ED peptide trigger the generation and nuclear import of PSA-NCAM by different pathways. (a–e) Cerebellar neurons were treated without (-) (a,b,e) or with chicken NCAM antibody (12.5 µg/ml IgY) (Ab) (b,c), ED peptide (25 µg/ml) (ED) (a,b,d,e) or ED control peptide (25 µg/ml) (ctrl) (a,b). (a) Nuclear fractions were isolated and subjected to immunoblot analysis with PSA antibody or with histone H3 antibody to control loading. (b) Cell lysates were probed by immunoblot analysis with an antibody against phosphorylated MARCKS (pMARCKS) or total MARCKS. Levels of phosphorylated MARCKS were quantified by densitometry and normalized to the levels of total MARCKS. Mean values with standard deviation from 3 independent experiments are shown for the levels of phosphorylated MARCKS in treated neurons relative to the levels in non-treated cells (set to 100%). (c,d) Cerebellar neurons were pretreated without (no inhibitor) or with inhibitors of FGF receptor (100 nM), calmodulin (10 µM), PLC (10 µM), PKC (1.5 µM), NOS (100 µM), FAK (10 µM), PLD (40 nM) or PI3K (4 µM) and then treated without (-) or with chicken NCAM antibody (12.5 µg/ml IgY) (Ab) (c) or ED peptide (25 µg/ml) (ED) (d) or were pretreated without (no inhibitor) or with inhibitors of MMP2 (25 nM) or MMP9 (10 nM) and then treated with the ED peptide (25 µg/ml) (e). Nuclear fractions were subjected to immunoblot analysis with the PSA-specific antibody or histone H3 antibody to control loading. Nuclear PSA levels were quantified by densitometry and normalized to the histone H3 levels. Mean values with standard deviation from 3 independent experiments are shown for the normalized PSA levels in treated cells relative to the PSA level of non-treated cells (set to 100%). (a,b,e) Representative immunoblot out of three experiments is shown. Lanes that were not adjacent to each other but derived from the same blot are indicated by dividing lines.

Fig 3: MDMX depletion inhibits UM cell growth and increases growth inhibition by Sotrastaurin treatment.a, c OMM2.3 and MEL202 i-shCtrl and i-shMDMX cells were incubated for 72 h with 10 ng/ml doxycycline, 0.5 µM Sotrastaurin or the combination before harvesting. The expression of MDMX and phosphorylated MARCKS was analyzed by western blot. Vinculin expression was analyzed to control for equal loading. b, d OMM2.3 and MEL202 i-shCtrl and i-shMDMX cells were seeded in quadruplicate in 12-well plates and incubated for 8 days with indicated compounds (OMM2.3: 20 ng/ml doxycycline and 0.5 µM Sotrastaurin; MEL202: 20 ng/ml doxycycline and 0.1 µM Sotrastaurin). Cell survival was determined using crystal violet staining. Data plotted are the normalized averages with the standard deviation as error bars. Combinations, which survival significantly differed compared with both single treatments, are indicated with an asterisk (*)

Fig 4: 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 PKCd 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 av 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 PKCd 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, PKCd 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 5: Effects of PMA on Npnt gene expression. (A) MC3T3-E1 cells were starved for 16 h in serum-free medium. Cells were treated with or without PMA (100 nM) for five minutes, then proteins were extracted and subjected to western blotting to detect phosphorylation of Marcks (p-Marcks) and actin. (B) MC3T3-E1 cells were treated with PMA (10 nM) for 24 h. Total cellular RNA was extracted, and mRNA levels of Npnt and Gapdh were examined using quantitative real-time PCR analysis. (C) Dose-dependent effects of PMA on Npnt expression. MC3T3-E1 cells were treated with PMA (0, 0.32, 1, 3.2, 10, or 32 nM) for 24 h and then examined using quantitative real-time PCR analysis. (D) Time course analysis of PMA effects on Npnt gene expression. MC3T3-E1 cells were treated with PMA (10 nM) for 0, 1, 3, 6, 12, or 24 h and then examined using quantitative real-time PCR analysis. Results are shown as the mean ± SD of 3 samples. **P < 0.01, Student’s t-test as compared to the level with 0 nM of PMA.