Foxo3a

The protein concentration in the hippocampus or cells was detected using a BCA kit (Biyuntian Institute of Biotechnology, China) and adjusted to the same level. The samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene PVDF membrane (Millipore, Bedford, MA, United States). After being blocked with 5% skim milk for 2 h, the membrane was incubated with primary antibodies overnight at 4°C and secondary antibodies for 2 h at room temperature. The specific primary antibodies were against SIRT1 (1:1,000, Proteintech Group, INC., China), FOXO3A (1:1,000, Proteintech Group, INC., China), and NLRP3 (1:1,000, Zen-Bioscience, China). The secondary antibodies were the horseradish enzyme-labeled goat antimouse secondary antibody (1:1,000, ZhongShan Jinqiao Biotechnology Co., Ltd., China) and the horseradish enzyme-labeled goat antirabbit secondary antibody (1:1,000, ZhongShan Jinqiao Biotechnology Co., Ltd., China). The protein bands on the membrane were recorded using a gel imaging system (UVP, California, United States).

Antibodies against the following proteins were used: p-p65 (sc136548, Santa cruz Biotechnology, Inc. USA) P-JNK (sc293136, Santa cruz Biotechnology, Inc. USA); P-ERK1/2 (sc81492, Santa cruz Biotechnology, Inc. USA); p-p38 (sc7973, Santa cruz Biotechnology, Inc. USA); Beclin1 (11306-1-AP, Proteintech, Wuhan, China); LC3A/B (AF5402, Affinity Biosciences, USA); p62 (55274-1-AP, Proteintech, Wuhan, China); FoxO3a (10849-1-AP, Proteintech, Wuhan, China); β-actin (60008-1-Ig, Proteintech, Wuhan, China); horseradish peroxidase (HRP) conjugated goat anti-mouse (ZDR-5109, ZSGB-BIO Biotechnology Co. Ltd. Beijing, China), and horseradish peroxidase (HRP) conjugated goat anti-rabbit (ZB-5301, ZSGB-BIO Biotechnology Co. Ltd. Beijing, China). The followings reagents were used: punicalagin (65995-63-3; Chengdu Herbpurify Co. Ltd.Chengdu, China); lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (L4391, Sigma-Aldrich, USA); chloroquine (C6628, Sigma-Aldrich, USA); and monodansylcadaverine (30432, Sigma-Aldrich, USA).

Tumor tissues were fixed in 4% paraformaldehyde for 24 h and were dehydrated through a serial concentrations of alcohol washes, embedded in paraffin and cut into 5-mm thick sections. Before immunostaining, the tissues were dewaxed in xylene and blocked with 10% sheep serum for 1 h at 37°C. The tissues sections were incubated with the KI-67, Bax, Bcl-2, cyclin B1, AMPK, or FOXO3A primary antibodies (Proteintech, United States, 1:200 dilution) overnight. The sections were then incubated with HRP labeled goat anti-rabbit IgG secondary antibody (Servicebio, Wuhan, China) for 1 h. Finally, the sections were stained with hematoxylin and images were captured by fluorescent microscopy using a Pannoramic 250 Flash III.

The skeletal muscle tissue (EDL) was homogenized using RIPA lysis buffer (Thermo, 89,900, IL, USA) supplemented with Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (Thermo, 1,861,280, IL, USA). The resulting supernatant was collected, and the total protein content was measured using the Rapid Gold BCA Protein Assay Kit (Thermo Scientific, A53226, IL, USA) according to the manufacturer’s instructions. Proteins (25 µg) were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels ranging from 8 to 12%. The separated proteins were then transferred onto polyvinylidene difluoride membranes (Bio-Rad, 1,620,177, CA, USA) and blocked with 5% skimmed milk (Bio-Rad, 1,706,404, CA, USA) for 2 h. Afterward, the membranes were washed three times with TBST and incubated overnight at 4 °C with primary antibodies. The primary antibodies used in this study included AKT, p-AKT (Ser473), mTOR, p70S6K, p-p70S6K, and p-FOXO3A (all from Cell Signaling Technology, MA, USA; 1:1000), as well as FOXO3A, FOXO1, NF-κB, and MuRF1 (all from Proteintech, Wuhan, China; 1:1000). A secondary antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Cell Signaling Technology, Danvers, MA, USA; 1:2000), was applied. The protein bands were visualized using the ChemiDoc system (Bio-Rad, California, USA) and quantified using Image J software.

Anti-MuRF1 and MuRF2 antibodies were used as described previously by Witt et al. and Hirner et al. [22 (link),23 (link)]. Akt, phospho Akt (Ser-473), FoxO3a, and phospho-FoxO3a (Ser-235) were commercially obtained: anti-Akt1 (pan; product #10176-2-AP), and anti-Phospho-Akt-Ser473 (product #664441-1) were from Proteintech (Proteintech Europe, Manchester, United Kingdom). Anti-FoxO3a (product PB9196) was from Boster (Pleasanton, CA, USA), and anti-FoxO3a-Phospho-Ser253 was from Abcam (product ab#154786, Cambridge, United Kingdom), respectively. For WB analysis, tissue lysates were prepared from snap-frozen tissues by powdering in a mortar. The homogenized powder was solubilized in 6-M urea buffer and separated on 4–10% PAGE essentially as described previously [19 (link),20 (link),21 (link)]. Blotted proteins were detected with alkaline phosphatase and quantified with AIDA software (Advanced Image Data Analyser, Version 2.11, for download see https://aidaimaging.com/download/), as described by Witt et al. and Hirner et al. [22 (link),23 (link)].

The brain tissues were fixed with 4% polyformaldehyde for 24 h. And then they were collected in 30% sucrose solution (S112228; Rhawn Co., Ltd.) for 48 h before being snapped free and cryosectioned at 20 μm thickness. Sections were incubated in 0.5% Triton X‐100 (T8200; Solarbio Co., Ltd.) for 20 min and blocked with PBS containing 3% goat serum (SL038; Solarbio Co., Ltd.) for 1 h at room temperature before permeating overnight at 4°C with primary antibodies. The brain tissues were then incubated with primary antibodies against IBA1 (019‐19741; Wako Pure Chemical Industries, Co., Ltd.), FoxO3a (66428‐1‐Ig; Proteintech Co., Ltd.), GPX4 (A11243; ABclonnal Co., Ltd.), LC3 (14600‐1‐AP; Proteintech Co., Ltd.), NeuN (26975‐1‐AP; Proteintech Co., Ltd.), 4‐HNE (bs‐6313R; Bioss Co., Ltd.) overnight at 4°C. After that, DyLight 549‐labelled goal anti‐mouse IgG (SA00013‐7; Proteintech Co., Ltd.) and DyLight 488‐labelled goal anti‐rabbit IgG (SA00003‐4; Proteintech Co., Ltd.) were added and incubated at 37°C for 1 h, and the nucleus was stained by DAPI (S2110; Solarbio Co., Ltd.). The cells were imaged using an inverted fluorescence microscope (DM IL, Leica Co., Ltd.).