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

Figure 4.

Figure 4

CREBH interacts with PPARα to regulate expression of LC3b in the liver upon being unfed. A) IP and Western blot analysis of interactions between CREBH and PPARα in the livers of mice under feeding or after being unfed for 6, 12, or 24 h. Total liver protein lysates pooled from 3 mice per group were immunoprecipitated with the anti-CREBH antibody, followed by immunoblotting with the anti-PPARα or anti-CREBH antibody. Levels of β-actin were determined by Western blot analysis as input controls. B, C) Luciferase reporter (Rep) analyses of transcriptional activation of the human LC3b (B) or Ulk1 (C) gene promoter by CREBH alone or in combination with PPARα. Hepa1-6 cells were transduced with the reporter vector or a vehicle. After 24 h, the transfected cells were infected with Ad expressing GFP (control), CREBH, or PPARα. A Renilla reporter plasmid was included in the cotransfection for the normalization of luciferase reporter activities. The same adenovirus titers were used for individual infections. Data represent means ± sem (n = 3 biologic repeats). Non-transf, nontransfected cell control. *P ≤ 0.05, **P ≤ 0.01. D) ChIP analysis of CREBH and PPARα binding activities to the LC3b, Atg7, Atg2b, and Ulk1 gene promoters in Huh-7 cells infected with Ad expressing GFP, the activated CREBH, PPARα, or the same titer combination of CREBH-expressing and PPARα-expressing Ad. An anti-CREBH (C) or anti-PPARα (P) antibody was used to pull down the exogenously expressed activated CREBH or PPARα binding complex from the chromatins isolated from the Huh-7 cells. PCR reactions were conducted using the primers amplifying the CRE or PPRE-binding motif present in the Lc3b, Atg7, Atg2b, or Ulk1 gene promoter. N, no antibody; I, input controls (nonimmunoprecipitated chromatin). E, F) Levels of the Lc3b (E), Atg7, Atg2b, and Ulk1 mRNAs (F) in livers of WT mice, CREBH-KO mice, or CREBH-KO mice infected with Ad expressing GFP or PPARα after being unfed for 14 h. G, H) Levels of the Lc3b (G), Atg7, Atg2b, and Ulk1 mRNAs (H) in livers of WT mice, PPARα-KO mice, or PPARα-KO mice infected with Ad expressing GFP or the activated form of CREBH after being unfed for 14 h. E–H) 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.