Figure S2.

APC protein undergoes LLPS. (A) Axin1 KO HEK293T cells (KO1) transfected with TopFlash-luciferase reporter and dAPC2 were treated with or without Wnt3a conditional medium for 24 h before harvesting for luciferase determination. The data are shown as mean ± SD (n = 3). Statistical analysis was performed with a two-tailed unpaired t test (***, P < 0.001). (B) Confocal fluorescence images showing colocalization of Axin1 and dAPC2 puncta in SW480 cells. (C) FRAP analysis of Axin1-mCherry protein puncta in SW480 cells with or without dAPC2-EGFP protein. (D) Differential interference contrast (DIC) images of droplets formed by different concentrations of dAPC2 after adding 10% PEG8000. (E) DIC images of the droplets formed by dAPC2 at different NaCl concentrations after adding 3.5% PEG8000. (F) DIC images of Axin1 droplets with or without the same concentration of dAPC2 protein (1 µM, 2.5 µM, and 5 µM) at 150 mM NaCl without a crowder. (G) Axin1 LLPS with or without dAPC2 in different concentrations without a crowder, as assessed by DIC imaging. (H) Turbidity of purified Axin1 with or without the same concentration of dAPC2 protein (0.5 µM, 1 µM, 2.5 µM, and 5 µM). Scale bar in B, 10 µm; in C–F, 2 µm. Vec, vector.