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

Figure 6.

Figure 6

CREBH regulates TFEB through PPARα in the liver under being unfed. A) Western blot analysis of TFEB proteins in the cytosolic and nuclear fractions from the livers of mice under feeding or after being unfed for 6, 12, or 24 h. Levels of β-actin and Lamin B1 were determined as loading controls for cytosolic and nuclear proteins, respectively. The graph shows the quantification of TFEB protein signals based on Western blot analysis. The intensity of TFEB protein signals, determined by Western blot densitometry, was normalized to that of input controls. Fold changes in protein levels were determined by comparison to one of WT control under the feeding condition. B–D) Expression levels of Tfeb (B), Pparα (C), and Pgc1α (D) 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 conditions. Data represent means ± sem (n = 3). *P ≤ 0.05, **P ≤ 0.01. E) ChIP analysis of CREBH and PPARα binding activities to the endogenous Tfeb gene promoter in the livers of CREBH-KO and WT mice under feeding or after being unfed for 12 h. For 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 Tfeb gene promoter. F) ChIP analysis of CREBH and PPARα binding activities to the Tfeb gene promoter in Huh-7 cells infected with Ad expressing GFP, the activated CREBH, PPARα, or the same titer of 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. PCR reactions were conducted using the primers amplifying the CRE or PPRE-binding motif present in the Tfeb gene promoter. N, no antibody; I, input controls. G) Levels of the Tfeb mRNA 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. H) Levels of the Tfeb mRNAs 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. For (G, 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.