Fig 2: Pfn1 depletion enhances PM recruitment of SHIP2.A, CRISPR-Cas9 genomic editing strategy of INPPL1 (SHIP2): a guide RNA–Cas9 protein complex and a single-stranded homology-directed repair (HDR) template is electroporated into HEK293A cells stably expressing NG1-10. Using the split fluorescent protein approach, this tags the SHIP2 protein with the 11th strand of the Neon Green (NG) beta barrel that assembles with the NG1-10 to form a working fluorescent tag. B, genomic confirmation of INPPL1 tagging. INPPL1-specific forward (Fwd) and reverse (Rvs) primers amplified NG11 carboxy-terminal knock-in region of the gene. C, representative TIRF microscopy images of 293A-NG2-1-10:INPPL1-NG11 pre-EGF and ∼3 min post-EGF stimulation. Single-particle PM recruitment measured by number of particles in defined region of interest (ROI) of 100 μm2 area (particle density). The particle density at baseline (five frames prestimulation) is subtracted to yield the change in density upon EGF stimulation. Three to four experimental replicates (34–35 cells total for each condition) presented as grand means ± SEM. INPP1, inositol polyphosphate 1-phosphate; Pfn1, profilin1; PM, plasma membrane; SHIP, SH2-containing inositol 5′ phosphatase; TIRF, total internal reflection fluorescence.

Fig 3: Effect of SHIP2 knockdown on focal adhesion formation and cell morphology in MDA‐MB‐231 cells. (a) Lysates from control (−) or SHIP2 siRNA‐transfected MDA‐MB‐231 cells were subjected to western blot analysis using antibodies against SHIP2, FAK, phospho‐FAK, Akt, phospho‐Akt (Ser‐473), PAX, phospho‐PAX, and actin. (b) MDA‐MB‐231 cells transfected with the indicated antibodies were stained with vinculin (VCL) and zyxin (ZYX) antibodies. F‐actin was visualized with Alexa Fluor 647 labeled‐phalloidin. Scale bar = 10 μm. (c) Quantification of focal adhesion number per cell from (b). (d) Measurement of cell area of cells from (b). All data represent the mean ± SD of 18 cells for the control, 16 cells for siSHIP2 #1, and 16 cells for siSHIP2 #2. *P < 0.05.

Fig 4: Evidence for Pfn1–SHIP2 interaction in cells. A and B, highest-ranked alpha-fold prediction of Pfn1’s interface with SHIP2 at its C-terminal polyproline-rich domain and the involved amino acid sequence for binding to Pfn1. C and D, representative images (panel C; the scale bar represents −10 μm) of proximity ligation assay to demonstrate endogenous Pfn1–SHIP2 interaction in MDA-231 cells with or without EGF stimulation (fluorescence images were intensity inverted for visual clarity of the PLA spots; dotted lines outline the cell periphery). As control, cells were transfected with Pfn1 siRNA (show a pronounced decrease in PLA spots) to demonstrate the specificity of interaction. Panel D quantifies EGF-induced changes in the average number of Pfn1:SHIP2 PLA spots/cell. Data are summarized from image analyses of five random 60× fields/experiment pooled from two independent experiments. Pfn1, profilin1; SHIP, SH2-containing inositol 5′ phosphatase.

Fig 5: Effect of INPPL1 siRNA on cell proliferation in HCT116 cells. Changes in proliferation in HCT116 cells 72 hours post-transfection with pooled control or pooled INPPL1 sequences. Fluorescence data is shown for cells only (no siRNA sequences), negative controls (non-silencing siRNA sequences), INPPL1 siRNA, and positive controls (siRNA sequences targeting essential genes). The effects of INPPL1 siRNA are based on differences between average fluorescence signal generated by negative controls and INPPL1 siRNA sequences. Asterisks denotes statistical significance.