Fig 2: Phosphorylation is critical to the functional interaction of PBF with CTTN. A, Representative 2D Boyden assay chamber cell invasion assay from three independent experiments showing TPC-1 cells transfected with VO, WT PBF, or Y174A PBF; representative photomicrographs are shown (i), as well as Western blots to confirm transfection (ii). Results are expressed as mean ± SEM. *, P < .05; **, P < .01. B, Representative 3D organotypic invasion assays in MDA-MB-231 cells transfected with VO, WT PBF or Y174A PBF. Box-whisper plots (right) show quantification of invasion area of MDA-MB-2331 cells. Statistics analyzed using Mann-Whitney U test. **, P < .01; ***, P < .001. C, Co-IP assays in HeLa cells transfected with VO, WT PBF, Y174A PBF, or CTTN as indicated and then treated with 50nM dasatinib (+) or vehicle (-). D, Quantification of scratch wound assay in MCF-7 cells stably transfected with VO, WT PBF, or Y174A PBF. Cells were treated with 50nM dasatanib (black) or vehicle (white) and percent wound recovery determined at the indicated time point postrecovery (h). Results are expressed as mean ± SEM. *, P < .05; **, P < .01; ***, P < .001. E, Schematic representation summarizing the proposed mechanism of PBF binding to CTTN and regulating tumor cell invasion. Proinvasive: (i) Src phosphorylates PBF and CTTN thereby promoting their interaction and actin filament assembly via downstream partners such as Arp2/3. Inhibitory: (ii) Dasatinib inhibits tyrosine phosphorylation by Src and diminishes binding between PBF and CTTN. (iii) Ablating phosphorylation at residue Y174 inactivates PBF and impairs the induction of tumor cell invasion.

Fig 3: Proinvasive effect of PBF and specific binding to CTTN. A, Representative 2D Boyden cell invasion assays from three independent experiments showing increased invasion of MCF-7 and TPC-1 cells transfected with PBF compared with VO. Results are mean ± SEM. Representative photomicrographs shown above. B, Representative 3D organotypic invasion assays in MDA-MB-231 cells transfected with VO or PBF. Box-whisper plots show quantification of invasion index and area of MDA-MB-231 cells in the presence (+) or absence (-) of cancer-associated fibroblasts. Statistics analyzed using Mann-Whitney U test. C, Representative images of scratch wound assay from three independent experiments in NIH3T3 cells stably transfected with VO or PBF at 4 and 6 h postrecovery with percent wound recovery shown (graph). Results are mean ± SEM. *, P < .05; **, P < .01; ***, P < .001. D, Representative Co-IP assays from three independent experiments showing specific interaction (*) between myc-tagged CTTN and HA-tagged PBF in MCF-7 and HeLa cells. E, Proximity ligation assays (PLA) assays showing specific interaction between PBF-HA and myc-CTTN in MCF-7, MDA-MB-231, and TPC-1 cells. Red fluorescent spots indicate specific interactions. White indicates DAPI nuclear staining. Magnification, 63×. Scale bars, 10 µM.

Fig 4: Involvement of Y174 in colocalization and binding of PBF with CTTN. A, Schematic of the protein domain structure of PBF showing the relative position of the YARF internalization motif. Boxed area highlights the alanine substitution at the Y174 residue in Y174A PBF. B, Representative immunofluorescence images showing subcellular colocalization (merge image, yellow) of endogenous CTTN (green) and endogenous pY174 PBF (red) in MCF-7 and HeLa cells. Intense “hot spots” of colocalization are outlined (yellow boxes). C, Confocal images showing colocalization (merge, yellow) between endogenous CTTN (green) and endogenous pY174 PBF (red) in MCF-7 cells. Framed areas in merge images are magnified and Pearson's colocalization coefficients are indicated. D, PLA assays showing specific interaction between endogenous CTTN and endogenous pY174 PBF in HeLa cells. Red fluorescent spots indicate specific interactions. Framed areas are magnified in lower panels. Scale bars, 10 µM. E, Representative Co-IP assays showing specific interaction between endogenous CTTN and endogenous pY174 PBF in MCF-7 and HeLa cells. F, Representative immunofluorescence images showing colocalization (merge, yellow) between myc-CTTN (green) and either WT PBF-HA (red) or Y174A PBF-HA (red) at the PM in SW1736 cells. Magnification, 100×. Scale bars, 20 µM. G, Co-IP assays showing increased binding (*) between myc-tagged CTTN and Y174A PBF vs WT PBF. Graph (right) shows quantification of PBF protein levels relative to ß-actin from three independent experiments in HeLa cells. Data expressed as mean PBF levels ± SEM. *, P < .05. H, PLA assays showing specific interaction between Y174A PBF and CTTN in TPC-1 and MD-MBA-231 cells vs WT PBF. Magnification, 63×. Scale bars, 10 µM. White indicates DAPI nuclear staining in panels B–D, F, and H.

Fig 5: Depletion of CTTN ablates the ability of PBF to induce cellular invasion. A, Representative photomicrographs of 2D Boyden assay chamber cell invasion experiments in SW1736, HCT116, and MCF-7 cells transfected with VO or PBF, and then treated with specific siRNAs for CTTN (siRNA 1 and 2) or control siRNA (CON). B, Quantification of number of invading cells per well from 2D Boyden assay described in panel A. Results expressed as mean ± SEM from three independent experiments. *, P < .05; **, P < .01; ***, P < .001. C, Western blot analysis of CTTN and PBF-HA expression in response to siRNA treatments as outlined in panel A. Graph (bottom) shows quantification of CTTN protein levels relative to ß-actin from siRNA experiments in SW1736 cells. Data presented as mean CTTN levels ± SEM from three independent experiments. *, P < .05; **, P < .01; ***, P < .001.