Figure 3

SIRT6 Cys144 displays lower activity than wild type and promotes pro-inflammatory glycolysis. (A) Cysteine residues coordinating the Zn-motif in Sirt6 are shown based on the 3PKI Sirt6 structure extracted from the Protein Data Bank. (B) SIRT6 activity assay was performed on cells transfected with wild-type SIRT6 plasmid or Cys144 mutant SIRT6 plasmid (n = 3). (C) Histone preparations were made on cells transfected with wild-type SIRT6 plasmid or Cys144 mutant SIRT6 plasmid and preparations were immunoblotted with H3K56 antibody (n = 3). (D) Cells transfected with wild-type SIRT6 or Cys144 mutant SIRT6 were stimulated with LPS for 3 and 6 hours and subjected to SIRT6 sulfenylation analysis (n = 3). (E–H) Effect of Cys144 mutation on Glycolysis in THP-1 cells: (E) Cells transfected with wild-type SIRT6 or Cys144 mutant SIRT6 were stimulated with LPS overnight. Media was collected for analysis of extracellular lactate production (n = 5). (F) Cells transfected with wild-type SIRT6 or Cys144 mutant SIRT6 were stimulated with LPS for 4.5 hours. Membrane fractions were made and evaluated for Glut-1 expression (n = 3). *P < 0.05 (two-tailed Student’s t-test for pairwise comparisons). (G,H) Cells transfected with wild-type SIRT6 or Cys144 mutant SIRT6 were stimulated with LPS for 6 hours and Glycolysis was assessed by Seahorse Respirometry (n = 4).