Fig. 6: SRT1720 suppresses AIF-mediated chromatinolysis and necrotic brain injury.
A, B SK-N-SH cells were pre-treated with DMSO or SIRT1 activator SRT1720 (10 µM) for 12 h as indicated. Then, the cells were untreated or treated with MNNG and stained 3’-OH DNA breaks (TUNEL, red) and analyzed using a fluorescence microscope. DAPI (blue) was used to visualize the nuclei. For MNNG treatment, cells were treated with 500 µM MNNG for 20 min, then cells were maintained in fresh medium for another 8 h. Representative overlay pictures are shown (A). Scale bars indicate 20 µm. And the frequency of TUNEL-positive labeling (% cell death) in (A) was counted and plotted as a percentage of total cells (B). The data represent the means of five independent experiments (n = 5) ± SD and are shown in each column. ***p < 0.01. C–E SK-N-SH cells were pre-treated with DMSO or the SIRT1-specific activator SRT1720 at a concentration of 10 µM for 12 h. Subsequently, the cells were untreated or exposed to MNNG. Following treatment, the cells were stained with Annexin V-FITC and propidium iodide (PI) and subsequently analyzed using flow cytometry 9 h after treatment. Representative cytofluorometric plots are presented (C). The percentage of cell death, indicated by Annexin V and PI-positive labeling, was quantified by flow cytometry and is depicted (D). The data represent the means ± standard deviation (SD) of five independent experiments (n = 5) and are displayed in each column. Statistical significance was determined, with *** indicating p < 0.01. The acetylation level of AIF was analyzed by western blot using an anti-AIF-K295ac antibody (E). F SK-N-SH cells were pre-treated with DMSO or the SIRT1-specific activator SRT1720 at a concentration of 10 µM for 12 h. Subsequently, the cells were untreated or exposed to MNNG. A CCK-8 assay was performed to measure cell viability as indicated time. The data represent the means of five independent experiments (n = 5) ± SD and are shown in each column. ***p < 0.01. G Effect of pharmacological modulation of SIRT1 on the infarct volume after MCAO was assessed. SIRT1 activator SRT1720 (200 mg/kg) dissolved in PBS or PBS (vehicle) was administered by intraperitoneal route 12 h before MCAO. The size of the MCAO-induced infarct was measured by triphenyl tetrazolium chloride (TTC) staining after MCAO (4 h). The infarct size was evaluated histomorphometrically on consecutive TTC-stained brain sections throughout the infarct (right panel). The data represent the means of five independent experiments (n = 5) ± SD, as shown in each column. ***p < 0.01. (H) DNA fragmentation was determined by gel electrophoresis in the brains of mice from sham and penumbra groups after MCAO treatment with or without SRT1720 pre-injection. I The acetylation levels of AIF-K295 in brain tissue samples after MCAO were analyzed by western blot using anti-AIF-K295ac and anti-AIF antibodies. J Immunoblots of nuclear translocation of AIF after MCAO. The post-nuclear (PN) and nuclear fractions (N) of samples after MCAO from (G) were prepared, α-tubulin (post-nuclear marker) and histone H3 (nuclear marker) were used to control fractionation quality and protein loading. The quantification of nuclear and post-nuclear AIF was conducted using ImageJ software. For the quantification of post-nuclear AIF, α-tubulin was used as a control, whereas Histone H3 served as the control for nuclear AIF quantification. K Model illustrating the role of AIF-K295 acetylation and the SIRT1-AIF axis in regulating chromatinolysis and caspase-independent programmed necrosis.
