Mnsod

Cells were lysed in RIPA buffer at 4°C for 30 min. Protein concentrations in the hPDLSCs were measured by a Pierce BCA Protein Assay Kit (Thermo Scientific, Pierce, Germany). A total of 40 μg of total protein from each sample was loaded onto an SDS-PAGE gel, and the proteins were separated by electrophoresis before being transferred onto PVDF membranes. After blocking in 5% nonfat milk, the membranes were incubated with primary antibody at 4°C overnight. Then, the membranes were washed three times with TBST buffer and incubated with HRP-linked antibody for 1 h at room temperature, followed by detection using the GeneGnome XRQ chemiluminescence imaging system. Primary antibodies against the following were used in this study: Runx2 (ABclonal, USA), SP7 (Abcam, UK), MnSOD (Cell Signaling, USA), catalase (Cell Signaling), FoxO1 (Cell Signaling), and β-actin (Sigma, USA).

The expression of manganese superoxide dismutase (MnSOD) (Cell Signaling Technologies, 1∶1000 dilution) was analyzed by standard Western blotting techniques. The band intensity of three independent experiments was measured using SigmaGel software and normalized to Actin (Cell Signaling Technologies, 1∶1000 dilution).

Extracts for western blot were homogenized in RIPA buffer. In brief, proteins were separated by electrophoresis and then transferred to polyvinylidene difluoride membranes, which were processed with primary antibodies. Next, we incubated these membranes in the appropriate diluted secondary antibody at room temperature for 1 h. The follow primary antibodies were used: p62 (1:1000; Abcam, catalog no. ab56416), LC3 (1:1000; Cell Signaling Technology, catalog no. 2775s), translocase of outer mitochondrial membrane 40 homolog (TOM40; 1:1000; Abcam, catalog no. ab51884), COXIV (1:1000; Cell Signaling Technology, catalog no. 11967), manganese superoxide dismutase (MnSOD; 1:1000; Cell Signaling Technology, catalog no. 13141), β-actin (1:1000; Cell Signaling Technology, catalog no. 3700), and NeuN (1:1000; Abcam, catalog no. ab104224).

Preparation of cell lysates and Western blotting was performed as described previously [28 (link)]. Antibodies for TFAM (transcription factor A, mitochondrial; aka TCF6; Cell Signaling Technology Cat# 8076S RRID:AB_10949110), SOD2 (manganese superoxide dismutase (MnSOD, Cell Signaling Technology Cat# 13141, RRID:AB_2636921)), CDK2 (Cell Signaling Technology Cat# 2546S; RRID:AB_2276129), cyclin E (Cell Signaling Technology Cat # 20808S), AMPKα (Cell Signaling Technology Cat# 5831S, RRID:AB_10622186), AMPKα (Thr172 (Cell Signaling Technology Cat# 2535S, RRID:AB_331250)), and GADPH (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated secondary antibody (Cell Signaling Technology Cat# 7074 RRID:AB_2099233) was purchased from Cell Signaling (Danvers, MA, USA). The immune complexes were detected by chemiluminescence and quantified using analyst/PC densitometry software (Bio-Rad Laboratories, Hercules, CA).

Cells were harvested, washed two times with ice-cold PBS and then resuspended in 20 mM Tris-HCl buffer (pH 7.4) containing 1% NP-40, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/mL aprotinin, 5 µg/mL pepstatin A, 1 µg/mL chymostatin, 5 mM Na₃VO₄ and 5 mM NaF. The cell lysate was centrifuged at 13,000× g for 20 min at 4 °C. Protein concentration was determined using the BCA assay (Sigma, St Louis, MO, USA). Proteins were separated by Tris-Glycine SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with antibodies, indicated as follows: p-Akt and Akt (1:1000, Cell Signaling Technology, Beverly, MA, USA); p-ERK and ERK (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); mTOR (1:500, Santa Cruz Biotechnology); Bax and Bcl-2 (1:500, Santa Cruz Biotechnology); catalase, Cu/Zn-SOD, Mn-SOD (1:1000, Cell Signaling Technology), and GAPDH (1:1000, Assay Designs, Ann Arbor, MI, USA). The membrane was exposed to X-ray film; protein bands were scanned and measured using ImageJ analysis software (version 1.37; Wayne Rasband, NIH, Bethesda, MD, USA), and normalized by GAPDH, an internal control.

Curcumin (purity > 98.0%) was obtained from National Institutes for Food and Drug Control (Beijing, China). Alcohol (Red Star wine, 56% v/v) was obtained from Beijing Red Star Co. Ltd. (Beijing, China). Detection kits for mitochondria SOD, GSH-Px, MDA, Na⁺/k⁺-ATPase, Ca²⁺-ATPase, and Ca²⁺/Mg²⁺-ATPase were purchased from the Jiancheng Institute of Biotechnology (Nanjing, China). Rhodamine 123 (Rh123) was purchased from Molecular Probes (Hayward, CA, USA). Protein lysate was purchased from Biyuntian Biotechnology Research (Shanghai, China). Total Protein Extraction Kit (P1250) and Nuclear Extraction Kit (P1200) were purchased from Applygen Technologies Inc. (Beijing, China). Primary anti-bodies of PGC-1α, NRF1, Mn-SOD, GRP78, PERK, p-PERK, IRE1α, p-IRE1α, IκBα, p-IκBα, NF-κB p65, and β-actin were from Cell Signaling Technology (Beverly, MA, USA). TNF-α, IL-1β, and IL-6 ELISA kits were purchased from Xinbosheng Co. Ltd (Shenzhen, China). All other chemicals were of analytical grade and were obtained from standard commercial suppliers.