(A) Mt1 gene promoter activity analysis by transfection of luciferase reporters and GFP, SIRT6, SIRT6 (H133Y), or Mtf1 plasmids in HEK 293T cells (n =6–7/group). (B) ChIP qPCR analysis of the association of SIRT6 and MTF1 with the proximal promoter of the human MT1A gene under the ethanol treatment condition in VL-17A (n =4/group). (C) Co-IP analysis of an interaction between SIRT6 and Mtf1 in HEK 293T cells. (D) Mtf1 deacetylation analysis by cotransfection of Mtf1 and SIRT6 or SIRT6 (H133Y) in VL-17A cells. (E) Dose-dependent Mtf1 deacetylation analysis in VL-17A cells. (F) Mtf1 acetylation analysis in SIRT6-deficient VL-17A cells. (G, H) Western blot and quantification analysis of SIRT6, MTF1, and MT1/2 proteins in the liver of normal controls and AH patients (n=6). (I) qPCR analysis of SIRT6, MTF1, MT1A, and MT2A mRNAs in the liver of normal controls and AH patients (n=6). Data are presented as mean ± S.E.M. In panels A and B: #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. GFP or PPIA; *p < 0.05, **p<0.01, and ***p<0.001 for the indicated promoter reporter assays. In panels H and I: #p < 0.05 and ##p < 0.01 for AH vs. Control. ACTN, actinin; AH, alcoholic hepatitis; ChIP, chromatin immunoprecipitation; Co-IP, co-immunoprecipitation; qPCR, quantitative PCR.