Compound c

Chromatographical acetonitrile was bought from Thermo Fisher Scientific (China). Analytical-grade petroleum ether (PE), methanol, and ethyl acetate were bought from Honeywell (USA). Ultrapure water was obtained from a Milli-Q system (Millipore, USA). The other chemicals were analysis-grade chemicals.
RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Logan, UT, USA). Cell counting kit 8 (CCK-8) was bought from DOJINDO (Japan). Antibodies (including β-actin, p-AMPK, AMPK, p-S6, S6, p-P70S6K, P70S6K, p-p53, p53, and COX-2) were purchased from Cell Signaling Technology (USA); Compound C (an AMPK inhibitor) was bought from Santa Cruz Biotech (USA). Matrix basement membrane was purchased from Corning Co., Ltd. (USA). The apoptosis-Hoechst Staining Kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Crystal violet reagent was purchased from Damao (Tianjin, China). Ki-67 was purchased from Wuhan Servicebio Technology Co., Ltd. The four PMFs obtained from CRCP were used in DMSO and were stored at −20°C and diluted for use.

For flow cytometric analysis of cellular senescence, cells were treated with EB-3D for 72 h and then the medium was replaced with fresh medium containing EB-3D or DMSO for the next 72 h. For rescue experiments, cells were pretreated with 2.5 μM of Compound C (Santa Cruz Biotechnology, Dallas, TX, USA) for 2 h and then treated with EB-3D for the next 3 days. Senescence was evaluated by flow cytometry on a Cytomics FC500 flow cytometer (Beckman Coulter) using di-β-D-galactopyranoside (C₁₂-FDG) (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), a fluorogenic substrate for β-gal activity, as previously described [49 (link)].

Antibodies directed against AMPKα1/α2, ACC, pSer⁷⁹-ACC were obtained from Cell Signaling Technology. Anti-AMPK α1 and anti-AMPK α2 antibodies were from R&D Systems. Anti p-Thr¹⁷²AMPK antibody and p-Thr¹⁷²AMPK blocking peptide were obtained from Santa Cruz Biotechnology24 (link). Anti-actin antibody was from BD Transduction Laboratory. Anti-MAP2, anti-α-tubulin and anti-acetylated-α-tubulin antibodies were from Sigma. Mouse monoclonal antibodies PHF1 (tau pSer³⁹⁶/Ser⁴⁰⁴)50 (link), CP13 (tau pSer²⁰²)51 (link), 2E12 or RZ3 (tau pThr²³¹)5 (link), DA9 (total tau aa102-140)36 (link), DA31 (total tau aa220-240)36 (link) and MC1 (conformation dependent antibody that recognizes only tau in a pathological conformation)52 (link) were previously described. 12E8 monoclonal antibody was obtained from Dr. Seubert (Elan Pharmaceuticals, San Francisco, CA; tau pSer^262/356)53 (link). As total tau antibody in the Western-blot experiments, we used tau Cter (homemade well characterized antibody recognizing the 11 amino-acids in the c-terminal part of tau27)54 (link). Tau5 (total tau) and AT180 (tau pThr231) were from Invitrogen.
AICAR, metformin, PD98059, H-89, LY294002 were purchased from Tocris; roscovitin and MARK/Par1 inhibitor from Merck; Compound C was from Santa-Cruz Biotechnology and rapamycin was from Cell Signaling Technology.