Sirt3

The livers of septic mice were collected and lysed with RIPA buffer. Total and nuclear proteins were extracted using the RIPA Lysis Buffer (Beyotime, Beijing, China) and a nuclear extraction kit (Beyotime, Beijing, China), respectively. Protein samples were separated using 10–12% SDS-PAGE and transferred onto PVDF membranes. Then, the membranes were washed and blocked for 120 min, followed by incubation with primary antibodies against NF-κB p-p65 (Abcam, USA), histone H3 (Affinity, China), Sirt3 (Abcam, USA), PGC-1α (Abcam, USA), SOD2 (Abcam, USA), acetylated SOD2 (Ac-SOD2) (Abcam, USA), caspase-1 (Abcam, USA), GSDMD (Abcam, USA), IL-1α (Abcam, USA), IL-1β (Abcam, USA), or GAPDH (Affinity, China) at 4°C overnight. All antibodies were diluted according to the manufacturers' instructions. The membranes were washed and incubated with secondary antibodies at room temperature for 1 h. Finally, the blots were detected using enhanced chemiluminescence substrate (ECL kit, Millipore). Signal intensities were measured using Fusion software.

HUVECs were seeded at a density of 1 × 10⁵ cells/mL, and were divided into three groups as described above (n = 3/group) when they were 80–90% confluent. After 4 hours of reoxygenation, radioimmunoprecipitation assay lysate (containing phenylmethylsulfonyl fluoride and a phosphorylase inhibitor) was added to each sample [30 (link)]. After being treated for 30 min on ice, the protein samples were collected and centrifuged at 4°C. The supernatants were obtained, and the protein concentration of each sample was determined using the bicinchoninic acid method [31 (link)]. The proteins were electrophoretically separated on gels and transferred to membranes. The membranes were blocked with 5% skimmed milk powder at room temperature for 2 hours. The gray values of the protein bands were determined using Image Pro Plus software. The gray value of the target protein was expressed relative to that of the internal reference [32 (link)]. The primary antibodies used in the present study were as follows: Bcl2 (1:1000, Cell Signaling Technology, #3498), Bax (1:1000, Cell Signaling Technology, #2772), caspase9 (1:1000, Cell Signaling Technology, #9504), c-IAP (1:1000, Cell Signaling Technology, #4952), GAPDH (1:1000, Cell Signaling Technology, #5174), Bad (:1,000; Abcam; #ab90435), Sirt3 (1:1000, Abcam, #ab86671), PGC-1α (1:1,000; Cell Signaling Technology, #2178).

RIPA lysis buffer (Beyotime, P0013B) was used to extract total protein from skeletal muscle of mice, and the mixture of the protease and phosphatase inhibitors (Beyotime, P1050) was added. The BCA protein concentration assay kit (Beyotime, P0012) was used to determine the protein concentration. The same number of protein samples was separated using 10% SDS-PAGE electrophoresis and transferred to 0.22 μm PVDF (Millipore, Billerica). Under the condition of room temperature, the PVDF membrane was blocked with 5% skim milk for 1 hour. GDDPH (1 : 1000, Abcam) and β-tubulin (1 : 1000, Abcam) were used as internal references, and c-JUN (1 : 800, Abcam), CHOP (1 : 1000, Abcam), HSP60 (1 : 2000, Abcam), CLPP (1 : 2000, Abcam), AKT (1 : 1500, Abcam), pAKT (1 : 1000, Abcam), SIRT3 (1 : 1000, Abcam), and MnSOD (1 : 2000, Abcam) were used as target proteins. The PVDF membrane sealed with milk was incubated with the primary antibody at 4°C overnight, followed by incubation with horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour. After adding Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore) in the dark chamber, the protein expression level was recorded by film and the protein grey value was analyzed using ImageJ software. The results were represented by the ratio of the target to an internal reference.

After treatments, the cells were fixed and blocked. Then, cells were incubated overnight at 4 °C with primary antibodies: SIRT1 (1/50), SIRT3 (1/50), 8-oxoguanine (8-oxoG) (1/50), and FOXO3 (1/100) (Abcam, Cambridge, UK). Following that, the secondary antibodies Alexa Fluor 546 and 488 (1/250; Molecular Probes, Eugene, OR, USA) were administrated to the cells. Subsequently, the nuclei cells were marked with DAPI (Sigma-Aldrich, San Luis, CA, USA). Cells were visualized on an Olympus BX51 microscope (Olympus Corporation, Hamburg, Germany), and the ImageJ program was used to quantify the fluorescence intensity of the micrographs.

Protein were extracted using RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitor cocktails (Roche, Germany). The supernatants were collected after centrifugation, and protein concentration was determined with bicinchoninic acid (BCA) kit (Beyotime, Haimen, China) according to the manufacturer's instructions. Then 30 μg of total protein was loaded into 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (PVDF, Millipore, Billerica, MA, USA), and 5% of the BSA in TBS containing 0.1% Tween-20 (TBS-T) was used to block the membrane at room temperature for 1 hour. Primary antibodies were added and incubated at 4°C for overnight. Bcl-2, p53, and cleaved caspase-3 antibodies were purchased from Cell Signaling (Danvers, MA, USA); SIRT3, SOD2, PHD2, and HIF-1α were obtained from Abcam (Cambridge, UK); GAPDH was from Protein Tech Group (Chicago, IL, USA) and used as an internal control. After primary antibody, the membranes were washed in TBS-T for three times and horse radish peroxidase-conjugated secondary antibodies (Protein Tech Group, Chicago, IL, USA) were added to the membranes at a dilution of 1: 5000 for 1 hour at room temperature. The density of immunoblotting bands was shown via enhanced chemiluminescent (ECL) substrate (Thermo Pierce, Rockford, IL, USA).