Fig. 5

Butyrate accelerates LRP5 mRNA decay by transactivating ARE binding protein ZFP36. a Stability of LRP5 mRNA in SK-BR-3 cells treated with NaBu (4 mM) or vehicle (n = 3 biological replicates). b Stability of LRP5 mRNA in MDA-MB-231 cells treated with 4 mM NaBu or vehicle (n = 3 biological replicates). c Relative luciferase activity of psiCHECK2-LRP5-3’UTR-WT, Mut1, Mut2, and Mut1 + 2 in HEK293T cells treated with NaBu (4 mM) or vehicle (n = 3 biological replicates). d Volcano plots displaying DEGs comparing SK-BR-3 cells treated with NaBu (4 mM) or vehicle. The number of significant variant genes (FC > 2, P < 0.05) are shown. e Relative ZFP36 and pre-ZFP36 mRNA levels of SK-BR-3 and MDA-MB-231 cells treated with NaBu for 48 h in dose-dependent manner (n = 3 biological replicates). f Relative ZFP36, LRP5, and β-catenin protein levels in SK-BR-3 cells after transfection with the siRNA targeting these three GPRs and treated with NaBu (4 mM). g Relative ZFP36, LRP5, β-catenin, Histone H3-K9, and Histone H3 protein levels in SK-BR-3 cells treated with NaBu (4 mM) and TSA (1 μM; MCE, HY-15144). h Genome browser images showing ChIP-seq signals for the ZFP36 promoter using data from the Cistrome Data Browse (GSM810671). i Relative fold change of the ZFP36 promoter binding motif in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle as analyzed by ChIP-qPCR (n = 3 biological replicates). j Enrichment of endogenous LRP5 3’UTR in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle was analyzed by RNA immunoprecipitation (RIP) (n = 3 biological replicates). Relative LRP5 and ZFP36 mRNA expression (k) and stability of LRP5 mRNA (l) in SK-BR-3 cells treated with knockdown of ZFP36 and NaBu (4 mM), as assessed by RT-qPCR (n = 3 biological replicates). m Relative luciferase activity of psiCHECK2-LRP5-3’UTR-WT, Mut1, Mut2, and Mut1 + 2 in HEK293T cells treated with ZFP36 knocked down and NaBu (4 mM) (n = 3 biological replicates). n Relative ZFP36, LRP5, p-GSK3β, GSK3β, and β-catenin protein levels in SK-BR-3 cells treated with ZFP36 knocked down and NaBu (4 mM). o Representative immunofluorescence images of β-catenin protein (green) in SK-BR-3 cells treated with ZFP36 knocked down and NaBu (4 mM). The nucleus was stained with DAPI (blue) (Scale bars, 25 μm). p ALDH⁺ SK-BR-3 cells treated with ZFP36 knocked down and NaBu (4 mM) (n = 3 biological replicates). Results are presented as mean ± s.d. (a–e, i, j) by a two-tailed, unpaired Student’s t-test, (k, l, m, p) by one-way ANOVA. For multiple comparisons, p-values were adjusted using the FDR correction. P values are as indicated