Leep2 complex regulates macropinocytosis. (A) TRITC-Dextran uptake in WT, leep2A⁻, leep2B⁻, and leep2A⁻leep2B⁻ (DKO) cells. (B) Quantification of TRITC-Dextran uptake, as shown in A. (C) Quantification of TRITC-Dextran uptake by flow cytometry analysis. (D) Generation time of WT and DKO cells. (E) Time-lapse imaging of macropinosome formation in WT and DKO cells. PHcrac-GFP was expressed to indicate the macropinocytic structures. Arrowheads point to macropinocytic cups that closed. (F) Quantification of the size of membrane ruffles. (G) Quantification of the rate of membrane ruffle formation. (H) Quantification of the rate of macropinosome formation. (I) Quantification of the size of nascent macropinosomes. (J) Quantification of random motility. Top: Cell trajectories. Bottom: Summary of motility parameters. (K) Quantification of chemotaxis along the folic acid gradient. Top: Cell trajectories. Bottom: Summary of chemotaxis parameters; FMI, forward migration index. The scatter plots show data points with means and SD; n represents the number of cells or events analyzed. The plot in D shows means and SEM; data were from three independent experiments. For J and K, the cell trajectories at the top were from one representative experiment, n represents the number of cells analyzed; data shown in the table were from three independent experiments, and the average of each biological replicate was used to calculate the means and SEM. Significance was determined by one-way ANOVA in B and by two-tailed unpaired t test in D, F, G, H, and I. Scale bars, 5 μm.