Fig 2: Effect of RWP on ACLY. (a) HEK293 cells were transiently transfected with pGL3 basic-LUC vectors containing the −3116/−20 bp full-length region of the ACLY gene promoter (3000) or a truncated version of this region (1000). Then, cells were triggered with LPS in the absence (LPS) or in the presence of RWP 20 μg/mL or RWP 200 μg/mL. Unstimulated cells were used as a negative control. The luciferase gene reporter activity was assessed after 24 hours. Primary human monocytes, preincubated for 1 hour with RWP, were activated to macrophages with LPS, and protein levels of ACLY (b) and acetylated H3 and total H3 (d) were evaluated. In (b, d) ACLY, acetylated H3, total H3, and β-actin proteins were immunodecorated with specific antibodies. The intensities of immunolabeled protein bands were measured by using a quantitative software and normalized to β-actin: values obtained are reported under western blot images. Protein expression levels in control sample were taken as 1, and other samples were expressed in the proportion of the control. (c) In cells treated as in (b, d) ACLY enzymatic activity was quantified. In (a) and (c), values represent means ± SD of three experiments with three replicates in each. Statistical analysis was performed by one-way ANOVA followed by Tukey's test for multiple comparisons. Different letters indicate significant differences at p < 0.05. C: control; L: LPS; R20: RWP 20 μg/mL; R200: RWP 200 μg/mL.

Fig 3: Adhesion and migration phenotypes of SIRT6-deficient cells and cells with ACLY inhibition. (A). Morphology of colonies formed by control or SIRT6 KO U2OS cells in soft agar colony formation assay. (B). Quantification of colonies with rough edge morphology. Colony counts were obtained from 10 random fields per well, four wells per cell type, n = 250, ** p < 0.01, two-tailed Student’s t-test. (C). Soft agar colony formation of control and SIRT6 KO HCT116 cells. Colony counts were obtained from three wells per cell type, * p < 0.05, two-tailed Student’s t-test. (D). Representative images of wound healing scratch assay in control and SIRT6 KO U2OS cells stably expressing empty vector (pFB), wild-type (WT), or H133Y mutant (HY) of SIRT6. Cells were grown to confluency then wounded, and wound closure was monitored every hour for 24 h. (E). Quantification of % wound closure after 24 h (Mean ± SEM of three independent experiments; * p < 0.05; ** p < 0.01; ns: not significant, two-tailed Student’s t-test). (F). Representative images of scratch assay in control and SIRT6 KO U2OS cells. Wound closure in the presence or absence of 50 µM of ACLY inhibitor (ACLYi) was followed over 24 h. (G). Quantification of % wound closure after 24 h (Mean ± SEM of three independent experiments, * p < 0.05, ** p < 0.01, two-tailed Student’s t-test). (H). Control or SIRT6 KO U2OS (left panel) and HCT116 (right panel) cell adhesion onto 1% fibronectin after 24 h treatment with vehicle or ACLY inhibitor (Mean ± SEM of three independent experiments, * p < 0.05, ** p < 0.01, two-tailed Student’s t-test).

Fig 4: ACLY level increases with SIRT6 depletion. (A). Increased ACLY protein levels in SIRT6-deficient U2OS cells generated by knockdown (KD) or Crispr/Cas9-mediated knockout (KO). (B). Increased ACLY protein expression in SIRT6 KO mice tissues with quantification (Mean ± SEM of three experiments, * p < 0.05, one-tailed Student’s t-test). (C). Sub-cellular fractionation showing increased levels of nuclear and chromatin ACLY in SIRT6 KO cells compared to controls. β-tubulin, fibrillarin, and Histone H3 are shown as markers for the cytoplasmic, nuclear, and chromatin-enriched fractions. (D). Increased ACLY in SIRT6 KO cells reversed by overexpression of SIRT6 WT but not SIRT6 HY mutant with quantification (Mean ± SEM of three experiments, * p < 0.05; ** p < 0.01; ns: not significant, one-tailed Student’s t-test). pFB, empty vector control. (E). Co-IP of ACLY with Flag-tagged wild-type (WT) and catalytically inactive SIRT6 proteins overexpressed in U2OS cells. The S56Y and H133Y mutations abolish SIRT6 catalytic activity, whereas R65A and G60A have partially impaired deacetylase and ADP-ribosyl transferase activities. The Histone H3 levels show chromatin association of the SIRT6 proteins. (F). Increased abundance of Flag-ACLY and endogenous ACLY protein in SIRT6 KO cells. (G). Relative nuclear Acetyl-CoA levels in control and SIRT6 KO cells, normalized to total protein (Mean ± SEM of seven experiments, two-tailed Student’s t-test).

Fig 5: Model for the interplay between SIRT6 and ACLY, with a feed-forward loop to repress gene expression by coordinately regulating both histone deacetylation and availability of acetyl-CoA for histone acetylation.