Fig. 4

Blockage of LRP5-mediated Wnt/β-catenin signaling via butyrate inhibits cancer stemness. a Heatmap showing differentially expressed genes (DEGs) between untreated SK-BR-3 cells and SK-BR-3 cells treated with 4 mM NaBu for 48 hours. The number of significant variant genes (FC > 2) are shown (FC, fold change) (n = 3 biological replicates). b Venn diagram showing the overlap of downregulated genes from NaBu-treated SK-BR-3 cells (FC > 2, P < 0.05, 1165 genes) and upregulated genes in MDA-MB-231 spheres (FC > 2, P < 0.05, 2571 genes) and the subsequent GO analysis. c Venn diagram showing the overlap of downregulated genes from NaBu-treated SK-BR-3 cells, upregulated genes in MDA-MB-231 spheres and stemness genes. d Expression of mRNA for the indicated genes in SK-BR-3 Ctrl cells and spheres (n = 3 biological replicates). e Relative LRP5 mRNA expression in SK-BR-3 cells treated with NaBu for 48 h in dose-dependent manner (n = 3 biological replicates). f Relative mRNA expression of LRP5, SOX2, and NANOG in Ctrl and LRP5-forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle (n = 3 biological replicates). g ALDH⁺ cells in Ctrl and LRP5-forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle (n = 3 biological replicates). h ELDA was performed in Ctrl and LRP5-forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle, and representative sphere images are shown (Scale bars, 50 μm). Stemness frequency with the upper and lower 95% confidence intervals that indicate the frequency of one stem cell in tumors. Spheres were counted from 24 replicate wells. i Representative images of spheroids formed by single cells in the 4 groups (Scale bars, 100 μm) (up), number of spheres per 1000 cells (d > 50 μm) (middle), and distribution pattern of sphere diameter (down). j Relative LRP5, p-GSK3β, GSK3β and β-catenin protein levels in Ctrl and LRP5-forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle. k Representative immunofluorescence images of β-catenin protein (green) in the 4 groups, with nuclear staining with DAPI (blue) (Scale bars, 25 μm). l Western blot detects the expression of nuclear and cytoplasmic protein extracts from Ctrl and LRP5 forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle. Actin was used as a cytoplasmic internal loading control and Lamin B1 as the nuclear internal control. Results are presented as mean ± s.d. (a, d, e) by a two-tailed, unpaired Student’s t-test, (f, g, i) by one-way ANOVA, h by the likelihood ratio test. For multiple comparisons, p-values were adjusted using the FDR correction. P values are as indicated