FIGURE 5:

Rac1 OE increases mesothelial clearance area of SKOV3ip spheroids. SKOV3ip cells Parental, GFP control, High-Rac1 OE, Rac1 CRISPR-Cas9 control, and Rac1 CRISPR-Cas9 KD clone 23 were seeded in U-bottom plates to form spheroids (100 cells/well). Spheroids were transferred to CellTracker Red–labeled monolayers of LP-9 mesothelial cells. Mesothelial cell clearance was imaged at 7 h and 24 or 25 h post–spheroid addition. Clearance areas were normalized at each time point by dividing the clearance area by the spheroid area at 1 h postaddition. (A, C, and E) Representative spheroid images at 1 h postaddition in bright field and mesothelial clearance areas at 7 and 25 h post–spheroid addition visualized using a TRITC filter. (B) Quantification of normalized mesothelial clearance area (MCA) of parental and High Rac1 OE cells at 25 h post–spheroid addition. Means ± SD (n = 18 in parental, and n = 23 in Rac1 High OE). (D) Quantification of normalized 25-h MCA comparing GFP and High-Rac1 OE cells. Means ± SD (n = 29 in GFP and n = 34 in High Rac1OE). (F) Quantification of normalized MCA at 25 h comparing Parental, Rac1 CRISPR-Cas9 control, and Rac1 CRISPR-Cas9 KD clone 23. Means ± SD (n = 27 in Parental, n = 23 in Rac1 CRISPR-Cas9 control, and n = 26 in Rac1 CRISPR-Cas9 KD clone 23). One-way ANOVA, followed by a Tukey’s test was performed for all three experiments. ****p < 0.0001. Scale bars are 500 μm.