FIGURE 2.

Transcription factor EB (TFEB) binds to DNM1L promoter and induces its gene expression in the liver. (A) Total liver lysates from healthy donors and patients with alcoholic hepatitis (AH) were subjected to western blot analysis. Note: DRP1 blot was from the same blot/samples from Figure 1A. (B) Correlation analysis of decreased dynamin-related protein 1 (DRP1) and TFEB protein expression from (A). (C) Primary human hepatocytes were treated with EtOH (80 mM), and total lysates were subjected to western blot analysis. (D) Male wild-type (WT) C57BL/6J mice (2–3 months old) were injected with Ad-null or Ad-Tfeb (5 × 10⁸ PFU per mouse, intravenously) followed by Gao-binge alcohol feeding. Total liver lysates and RNA were subjected to western blot analysis and (E) qPCR analysis. (F) Primary mouse hepatocytes were infected with Ad-Tfeb or Ad-shRNA-Tfeb (G) for indicated dose and time. Total cell lysates were subjected to western blot analysis. (H) Primary mouse hepatocytes were infected with the indicated dose of Ad-Tfeb for 24 h, followed by the transfection of a luciferase reporter plasmid containing around 2000 bp mouse Dnm1l gene promoter sequence. Luciferase intensity was detected at 48 h from seeding. (I) Chromatin immunoprecipitation assay for the binding of TFEB to the Dmn1l promoter was performed in WT mouse livers. All results are expressed as means ± SEM (n = 3–6). *p < 0.05, **p < 0.01, ***p < 0.001; one-way analysis of variance analysis with Bonferroni’s post hoc test for (E), two-tailed Student’s t test for (H) and (I). Ad-, adenovirus-; EtOH, ethanol; IP, immunoprecipitation; MOI, multiplicity of infection; NC, nect qRCR, real-time quantitative PCR; shRNA, short hairpin RNA.