Fig 1: Sirt6 knockdown partially reverses the inhibitory effect of luteolin on TNF-α-induced inflammatory damage of HNPCs. (A and B) The Sirt6 protein (A) and mRNA (B) expression levels were detected by western blot analysis and RT-qPCR, respectively. (C-E) Effects of Sirt6 knockdown on cell viability (C) and intracellular IL-1β (D) and IL-6 (E) expression levels. (F) TUNEL staining was used to detect the effect of Sirt6 knockdown on HNPC apoptosis. (G) Expression levels of Bcl-2, Bax and cleaved caspase 3 protein were detected by western blot analysis. ***P<0.001 vs. control or si-NC. ##P<0.01 and ###P<0.001 vs. TNF-α. +P<0.05 ++P<0.01 and +++P<0.001 vs. TNF-α + luteolin. @@P<0.01 and @@@P<0.001 vs. TNF-α + luteolin + si-NC. TNF-α, tumor necrosis factor-α; HNPCs, human nucleus pulposus cells; Sirt6, sirtuin 6.
Fig 2: Luteolin regulates the Sirt6/NF-κB pathway. (A) The expression levels of Sirt6/NF-κB pathway-related proteins (Sirt6, p-NF-κB and p-NF-κB p65) were examined by western blot analysis. (B) Western blot analysis was used to examine histone acetylation-related H3K9ac expression level. (C) Effect of luteolin on TNF-α-induced Sirt6 activity was detected by SIRT6 activity assay kit. (D) Interaction between Sirt6 and luteolin was predicted by molecular docking. ***P<0.001 vs. control. #P<0.05, ##P<0.01 and ###P<0.001 vs. TNF-α. TNF-α, tumor necrosis factor-α; HNPCs, human nucleus pulposus cells.
Fig 3: DMW modulates SIRT6 and SIRT3 expression and activity in xenograft model of colorectal cancer. Representative immunofluorescence images and analysis, protein expression levels and deacetylase enzymatic activity of (A–D) SIRT6 and (E–H) SIRT3 in control and DMW-treated mice. Fluorescence intensity determination, reported as boxplots representing the densitometric mean values of arbitrary fluorescence units (AFU). For immunoblotting study, boxplots represent the densitometric mean values, expressed as arbitrary units (AU) of n = 5 different experiments. Protein expression was determined after normalisation with α-tubulin as internal control with ImageJ software. Lane 1 = molecular marker; Lanes 2, 3, and 4 = control mice; Lanes 5, 6, and 7 = DMW-treated mice. ** p < 0.01 and *** p < 0.001 vs. control mice.
Fig 4: Knockdown of Sirt6 partially reverses the inhibitory effect of luteolin on TNF-α-induced senescence of HNPCs. (A) The activity level of β-galactosidase in HNPCs was assayed by senescence β-galactosidase staining. (B) ELISA kit was performed to detect the activity of telomerase. (C) Expression levels of senescence-related proteins (p16 and p21) were examined via western blot analysis. (D) Expression of p53 was detected by western blot analysis. ***P<0.001 vs. control. ###P<0.001 vs. TNF-α. +++P<0.001 vs. TNF-α + luteolin. @@P<0.01, @@@P<0.001 vs. TNF-α + luteolin + si-NC. TNF-α, tumor necrosis factor-α; HNPCs, human nucleus pulposus cells; Sirt6, sirtuin 6.
Fig 5: Smooth muscle LOX-1 deficiency ameliorates atherosclerosis in LKB1SMKO mice.CTR and LKB1SMKO mice were injected with AAV9-shCON or AAV9-shLOX-1 1 week before injection with rAAV8/D377Y-mPCSK9 and a Paigen diet feeding for 12 weeks. A Oil-red O staining in aortas (n = 6). *P < 0.05, **P < 0.01 vs CTR + AAV9-shCON, ##P < 0.01 vs LKB1SMKO + AAV9-shCON. B Hematoxylin and eosin (H&E) staining in aortic roots (n = 6). Scale bar = 200 μm. *P < 0.05, ***P < 0.001 vs CTR + AAV9-shCON, ###P < 0.001 vs LKB1SMKO + AAV9-shCON. C Oil-red O staining in aortic roots (n =6). Scale bar = 200 μm. *P < 0.05, ***P < 0.001 vs CTR + AAV9-shCON, ###P < 0.001 vs LKB1SMKO+AAV9-shCON. D Immunohistochemical staining of MOMA-2 in aortic roots (n = 6). Scale bar = 200 μm. *P < 0.05, **P < 0.01 vs CTR + AAV9-shCON, ##P < 0.01 vs LKB1SMKO + AAV9-shCON. E Double labeling immunofluorescent staining of BODIPY and α-SMA in aortic roots (n = 6). Scale bar = 100 μm. *P < 0.05, **P < 0.01 vs CTR + AAV9-shCON, #P < 0.05 vs LKB1SMKO + AAV9-shCON. F Levels of TC, TG, HDL-C, and LDL-C in serum. Data were analyzed by one-way ANOVA followed by Bonferroni multiple comparison analysis (A–F). G Smooth muscle LKB1 inhibits VSMC-derived foam cell formation and atherosclerosis via phosphorylation of SIRT6 and subsequent inhibition of LOX-1 expression.
Supplier Page from Abcam for SIRT6 Activity Assay Kit (Fluorometric)