Fig 2: PAA activates Sirt3 during renal fibroblast activation and interstitial fibrosis.a The chemical structure of PAA. b Binding models of PAA (pink) in Sirt3 (white). c The interaction of PAA with the amino acid of Sirt3. d Masson’s trichrome staining of kidney tissues of UUO rats at 2nd week. Magnification, ×100. e The mRNA expression of Sirt3 in UUO rats. f, g The protein expression and relative quantitative data of Sirt3 in UUO rats. h Immunofluorescent staining of vimentin and relative quantitative data in NRK-49F cells after 24 h treatment. Scale bar, 50 μm. i The cell viability of NRK-49F cells after 24 h treatment. j The mRNA expression of Sirt3 in NRK-49F cells. k, l The protein expression and relative quantitative data of Sirt3 in NRK-49F cells. Data were presented as mean ± SD. Each dot presented the single data result in bar graph. **P < 0.01 vs sham-operated or control group (n = 6). #P < 0.05, ##P < 0.01 vs UUO or TGF-β1 group (n = 6)

Fig 3: HKL protects vitiligo melanocytes against oxidative stress by activating SIRT3-OPA1 axis. (A-B) PIG3V cells were pre-treated with 5 μM HKL for 24 h and then treated with 1.0 mM H2O2 for 24 h. The mRNA and the protein levels of SIRT3 were detected by qRT-PCR and immunoblotting. (C) SIRT3 activity of PIG3V cells with treatment as indicated was measured based on an enzymatic reaction using a SIRT3 activity assay kit. Data represent mean ± SD (n = 3). (D) The protein level of SOD2 and Ac-SOD2 in PIG3V cells with treatment as indicated were detected. Mean ± SD is shown (n = 3). (E) Representative confocal microscope images of the mitochondrial network in PIG3V cells with treatment as indicated. Scale bar = 50 μm (Magnification: the upper = 600 ×; the under = 1800 ×). The proportion of PIG3V cells (n=100 cells for each sample) with tubulated, intermediate and fragmented mitochondria was quantified. (F) The apoptosis level of PIG3V cells with treatment as indicated was examined by annexin V-FITC/PI staining. The mitochondrial ROS level of PIG3V cells with treatment as indicated was examined by MitoSOX™ Red mitochondrial superoxide indicator staining. The mitochondrial membrane potential level of PIG3V cells with treatment as indicated was analyzed by JC-1 staining. Assessment of ATP level in PIG3V cells with treatment as indicated. Data represent mean ± SD of triplicates. (G) Representative confocal microscope images of the mitochondrial network in PIG3V cells with treatment as indicated. si-NC refers to negative control small interfering RNA, si-OPA1 refers to small interfering RNA against OPA1. Scale bar = 50 μm (Magnification: the upper = 600 ×; the under = 1800 ×). The proportion of PIG3V cells (n=100 cells for each sample) with tubulated, intermediate and fragmented mitochondria was quantified. (H) The apoptosis level of PIG3V cells with treatment as indicated was examined by annexin V-FITC/PI staining. The mitochondrial ROS level of PIG3V cells with treatment as indicated was examined by MitoSOX™ Red mitochondrial superoxide indicator staining. The mitochondrial membrane potential level of PIG3V cells with treatment as indicated was analyzed by JC-1 staining. Assessment of ATP level in PIG3V cells with treatment as indicated. Data represent mean ± SD of triplicates.(I) The level of apoptosis-related proteins in PIG3V cells with treatment as indicated was detected by immunoblotting. (J) The level of cytochrome c in mitochondria or cytoplasm in PIG3V cells with treatment as indicated was detected by western blotting. TOMM20 was detected as loading control for mitochondrial fraction. Tubulin was detected as loading control for cytosolic fraction. β-Actin was detected as loading control for the whole cell lysate. (K) Relative enzyme activity of respiratory complexes I, II, III, IV and V were measured in PIG3V cells with treatment as indicated. Data represent mean ± SD of triplicates. p value was calculated by two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: CYP inhibition causes a selective decrease in apoptotic proteins and elevated SIRT3 levels in human EPI-ECs and neuronal cells. (A,B) Western blot showed a significant decrease in the BAX (pro-apoptotic)/Bcl-XL (anti-apoptotic) ratio in LCM + OXC (*p < 0.05) and LCM (*p < 0.05) expression in EPI-ECs (A) and in LCM + OXC (***p < 0.001) and LCM (*p < 0.05) expression in neuronal cells (B), post-CYP inhibition with ketoconazole (KCZ) within the same cell types. Similarly, the levels of ERK1/2 (*p < 0.05), phospho-ERK (***p < 0.001, LCM + OXC and *p < 0.05, LCM), caspase-3 (*p < 0.001) and cytochrome c (***p < 0.001) showed a significant decrease post-CYP inhibition, markedly in LCM + OXC co-treated and LCM alone group in EPI-ECs along with improved SIRT3 (***p < 0.001) levels. Likewise in neuronal cells (B), a similar pattern was seen with significantly decreased levels of ERK1/2 (**p < 0.01), phospho-ERK (*p < 0.001) and cytochrome c (*p < 0.001) post-CYP inhibition in the LCM + OXC co-treated group and increased SIRT3 (*p < 0.001) levels. Western blot quantitative data (in right) are normalized with β-actin, and values are plotted as mean ± SEM by two-way ANOVA with a Tukey post hoc test for each target, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: 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.