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. 2019 Mar 26;33(7):7896–7914. doi: 10.1096/fj.201802528R

Figure 3.

Figure 3

CREBH directly regulates expression of the key autophagy genes in the liver in response to being unfed. A) Expression levels of Lc3b, Atg7, Atg2b, and Ulk1 mRNAs in the livers of CREBH-KO and WT mice under feeding conditions or after being unfed for 6, 12, or 24 h. mRNA expression levels were determined by qPCR. Fold changes in mRNA levels were determined by comparison to the mRNA levels in one of the WT control mice under the feeding condition. Data represent means ± sem (n = 3). *P ≤ 0.05, **P ≤ 0.01. B) Expression levels of Atg7, Atg2b, Ulk1, and Lc3b mRNAs in the livers of mice infected with Adenovirus (Ad) overexpressing GFP or activated CREBH. mRNA expression levels were determined by qPCR. Fold changes in mRNA levels were determined. Data represent means ± sem (n = 3). *P ≤ 0.05, **P ≤ 0.01. C) Western blot analysis of CREBH, LC3b, ATG7, ATG2b, P62, and Ulk1 proteins in the livers of CREBH-KO and WT mice under feeding conditions or after being unfed for 6, 12, or 24 h. Levels of β-actin were determined as loading controls. CREBH (P), CREBH precursor; CREBH (A), activated/cleaved form of CREBH; LC3-I, a cytosolic form of LC3b; LC3-II, lipid-modified LC3b form. D) Western blot analysis of CREBH, LC3b, ATG7, ATG2b, and Ulk1 proteins in the livers of CREBH-KO and WT mice infected with Ad overexpressing GFP or activated CREBH. E) Binding activities of mouse liver NEs to the CRE-binding motif of the mouse Lc3 gene promoter. NEs were prepared from the livers of CREBH-KO and WT control mice under feeding or after being unfed for 6, 12, or 24 h. EMSA was performed with liver NEs and biotin-labeled Lc3 gene probe containing the CRE-binding motif. F) Binding activities of mouse liver NE expressing GFP, CREBH, or the CREBH mutant K294R to the CRE-binding motif of the mouse Lc3b gene. NE was prepared from the livers of WT mice infected with Ad expressing GFP, CREBH (activated form), or K294R. EMSA was performed using the liver NEs and biotin-labeled Lc3b gene probe containing the CRE-binding motif. G) ChIP analysis of CREBH- and PPARα-binding activities to the endogenous Lc3b, Atg7, Atg2b, and Ulk1 gene promoters in the livers of CREBH-KO and WT mice under feeding or after being unfed for 12 h. For ChIP-PCR, isolated mouse chromatins were immunoprecipitated with the antibody against CREBH (C), PPARα (P), or no antibody (N). Nonimmunoprecipitated chromatins were included as input controls (I). PCR reactions were conducted using the primers amplifying the CRE or PPRE-binding motif present in the Lc3b, Atg7, Atg2b, or Ulk1 gene promoter. The amplified PCR products were visualized on agarose gels.