Figure 5.
Figure 5. Disrupting ciliogenesis activates the MVA pathway by positively regulating β-catenin–TCF signaling. (A) Lithium elevated the mRNA level of enzymes in the MVA pathway. MEF cells were treated with 25 mM LiCl, and the mRNA levels of MVA enzymes were examined using real-time PCR. (B) Knocking down the expression of β-catenin reduced the level of the mRNA levels of Hmgcs, Hmgcr, Idi1, and Cyp51 proteins in MEFs and NIH3T3 cells. (C) ChIP assay using β-catenin antibody to examine the interaction between β-catenin and promoters of HMGCS, HMGCR, IDI1, CYP51, and mouse Hmgcr. Real-time PCR was performed. (D and E) dCas9-edited HMGCR promoter (dCas9 2 and dCas9 5) impaired the expression of HMGCR mRNA (D) and protein level (E) induced by overexpressing β-catenin. The TBE2 in the endogenous HMGCR promoter of HPDE6C7 cells was edited by the dCas9 system. The details are described in Materials and methods. (F) dCas9-edited HMGCR promoter (dCas9 2 and dCas9 5) impaired its binding with β-catenin. The complex of β-catenin and DNA was immunoprecipitated using β-catenin antibody. Real-time PCR was performed to examine the TBE2 of HMGCR promoter. (G) Mutation of TBE2 (mTBE2) impaired the activation of HMGCR promoter synchronized by β-catenin and SREBP2N. The Flag-tagged N terminus of SREBP2 (Flag-SREBP2N) plasmid and the myc-tagged β-catenin (myc–β-catenin) plasmid were transfected with human HMGCR promoter (WT) or the HMGCR promoter with TBE2 mutation (mTBE2) into 293T cells. The activities of the reporters were measured. (H) GST pull-down assay and immunoprecipitation assay were performed to show the interaction between β-catenin and SREBP2N. (I) dCas9-edited HMGCR promoter (dCas9 2 and dCas9 5) impaired the interaction between SREBP2 and TBE2 in the HMGCR promoter. The complex of SREBP2 and DNA was immunoprecipitated using SREBP2 antibody. Real-time PCR was performed to examine the TBE2 of HMGCR promoter. (J) dCas9-edited TBE2 in the HMGCR promoter impaired the increased HMGCR mRNA level synchronized by LiCl and SREBP2N. (K) ICAT and dominant-negative β-catenin impaired the induction of Hmgcs1, Hmgcr, Cyp51, and Idi1 after disrupted ciliogenesis. Student’s t test was performed. ##, P < 0.01; *, P < 0.05; **, P < 0.01. The data are presented as the mean ± SEM. Representative data are of at least two independent experiments. si con, siRNA control.

Disrupting ciliogenesis activates the MVA pathway by positively regulating β-catenin–TCF signaling. (A) Lithium elevated the mRNA level of enzymes in the MVA pathway. MEF cells were treated with 25 mM LiCl, and the mRNA levels of MVA enzymes were examined using real-time PCR. (B) Knocking down the expression of β-catenin reduced the level of the mRNA levels of Hmgcs, Hmgcr, Idi1, and Cyp51 proteins in MEFs and NIH3T3 cells. (C) ChIP assay using β-catenin antibody to examine the interaction between β-catenin and promoters of HMGCS, HMGCR, IDI1, CYP51, and mouse Hmgcr. Real-time PCR was performed. (D and E) dCas9-edited HMGCR promoter (dCas9 2 and dCas9 5) impaired the expression of HMGCR mRNA (D) and protein level (E) induced by overexpressing β-catenin. The TBE2 in the endogenous HMGCR promoter of HPDE6C7 cells was edited by the dCas9 system. The details are described in Materials and methods. (F) dCas9-edited HMGCR promoter (dCas9 2 and dCas9 5) impaired its binding with β-catenin. The complex of β-catenin and DNA was immunoprecipitated using β-catenin antibody. Real-time PCR was performed to examine the TBE2 of HMGCR promoter. (G) Mutation of TBE2 (mTBE2) impaired the activation of HMGCR promoter synchronized by β-catenin and SREBP2N. The Flag-tagged N terminus of SREBP2 (Flag-SREBP2N) plasmid and the myc-tagged β-catenin (myc–β-catenin) plasmid were transfected with human HMGCR promoter (WT) or the HMGCR promoter with TBE2 mutation (mTBE2) into 293T cells. The activities of the reporters were measured. (H) GST pull-down assay and immunoprecipitation assay were performed to show the interaction between β-catenin and SREBP2N. (I) dCas9-edited HMGCR promoter (dCas9 2 and dCas9 5) impaired the interaction between SREBP2 and TBE2 in the HMGCR promoter. The complex of SREBP2 and DNA was immunoprecipitated using SREBP2 antibody. Real-time PCR was performed to examine the TBE2 of HMGCR promoter. (J) dCas9-edited TBE2 in the HMGCR promoter impaired the increased HMGCR mRNA level synchronized by LiCl and SREBP2N. (K) ICAT and dominant-negative β-catenin impaired the induction of Hmgcs1, Hmgcr, Cyp51, and Idi1 after disrupted ciliogenesis. Student’s t test was performed. ##, P < 0.01; *, P < 0.05; **, P < 0.01. The data are presented as the mean ± SEM. Representative data are of at least two independent experiments. si con, siRNA control.

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