Figure 3. MYCBP2 CRISPR HeLa cells exhibit reduced ephrin-B2 evoked cell retraction and ephrin-B2 stripe avoidance.

(A) Schematic of generation of stable CTRL^CRISPR or MYCBP2^CRISPR HeLa EPHB2-FLAG cells. Note that these are not clonal cell lines. (B) MYCBP2 is reduced in HeLa MYCBP2^CRISPR cells generated by sgRNA targeting MYCBP2 exon 6. (C) Representative images of cell collapse assays using CTRL^CRISPR or MYCBP2^CRISPR HeLa cells that were stimulated with ephrin-B2. Red arrows indicate rounded/collapsed cells. Scale bar is 10 μm, (D) Quantification of collapsed cells. Statistical significance between CTRL^CRISPR and MYCBP2^CRISPR cells was determined using two-way ANOVA followed by Sidak’s multiple comparison test (CTRL^CRISPR vs MYCBP2 ^CRISPR: Fc, p=0.9903; eB2-Fc, p<0.0001. Fc vs eB2-Fc: CTRL^CRISPR, p<0.0001; MYCBP2 ^CRISPR, p=0.0003). (E) Representative time-lapse sequences of CTRL^CRISPR and MYCBP2^CRISPR HeLa cells after ephrin-B2 treatment. (F) Quantification of cell area reduction after 60 min exposure to ephrin-B2. Cell area contraction ratio: CTRL^CRISPR, 27.1%; MYCBP2^CRISPR, 18.8%. p=0.0268, two-tailed unpaired t test. Data points corresponding to cells in representative images in panel E are in blue. (G) Ephrin-B2 stripe assays using CTRL^CRISPR or MYCBP2^CRISPR HeLa cells. Cells are visualized with Phalloidin 488 staining and nuclei are stained with DAPI (black, Fc stripes; red, ephrin-B2 stripes). Scale bar is 50 μm. (H) Quantification of cells present on ephrin-B2 stripes (%). Statistical significance was determined using two-tailed unpaired t-test (p=0.0109). Error bars represent SD.