Fig. 3: Identifying Rosmanol quinone (RQ) as a condensate-inducing therapeutic (c-inds) of β-catenin.

a–c Fluorescence recovery after photobleaching (FRAP) of EGFP-fused β-catenin condensates in U293T cells was analyzed. Data were collected from three independent experiments. a Representative time-lapse microscopy images show the effects of 0, 5, and 10 μM RQ on β-catenin condensates after 6-h incubation. Scale bars: 2 μm. Bleached areas are marked with blue circles. b The number of β-catenin condensates was quantified in ten randomly selected cells per sample. c Mean fluorescence intensity plots represent FRAP results for three condensates per condition (error bars: mean ± SD). d Fluorescence intensity analysis of green, fluorescent β-catenin in cytoplasm and nucleus in response to the 6-h incubation with 0, 5, and 10 μM RQ. The number of β-catenin levels was quantified in a random selection of 30 cells from each sample. e Schematic diagram of RQ-induced LLPS of β-catenin. RQ can induce liquid-liquid phase separation (LLPS) of cytoplasmic β-catenin, resulting in physical interphase blocking and subsequent inhibition of nuclear translocation. f The Western blot analysis was performed to examine the expression levels of β-catenin (β-cat), active β-catenin (Aβ-cat), c-myc (MYC), and Cyclin D1 (CTND1) in three Wnt-hyperactivated HCC cell lines, Hep3B, HepG2, and Hepa1-6, after treatment with 10 μM RQ for 24 h. n = 3 biological replicates. Data were presented as mean values ± SD. g The cell viability of three Wnt-hyperactivated HCC cell lines, Hep3B, HepG2, and Hepa1-6, was assessed after 48 h of treatment with gradient-diluted RQ. The median inhibitory concentrations (IC50) were determined by averaging the results from four independent experiments (n = 4). Data were presented as mean values ± SD. All box-whisker plots B, D center on the median; the bounds of the boxes mark the upper and lower quartile; the whiskers extend to the upper and lower extremes (1.5x interquartile range from the upper and lower quartiles). The p < 0.001 in b, d represents p = 10E-15. p value in b, d calculated by two-tailed t-test.