Sto 609

Ang II, N-acetyl-L-cysteine (ROS scavenger), irbesartan (angiotensin receptor blocker), STO-609 (CaMKK inhibitor), compound C (AMPK inhibitor), KN-93 (CaMKII inhibitor), fluo-3 AM, 2′,7′-dichlorofluorescein diacetate (DCFDA), L-ascorbic acid, Claycomb medium, norepinephrine, gelatine, fibronectin, and monoclonal anti-β-actin antibody were purchased from Sigma Chemical Company (St. Louis, MO, USA). Monoclonal antibodies against ACC, CaMKII and CaMKIIδ and polyclonal antibodies against AT1R, TGF-β1, phosphorylated ACC, and phosphorylated CaMKII were purchased from Abcam (Cambridge, MA, USA). Monoclonal antibodies against NF-κB, phosphorylated JNK and AMPKα, and polyclonal antibodies against JNK and AMPKα were obtained from Cell Signaling Technology (Danvers, MA, USA). Foetal bovine serum (FBS), L-glutamine, and penicillin-streptomycin were acquired from Thermo Fisher Scientific (Foster City, CA, USA).

cis-5,8,11,14,17-Eicosapentaenoic acid (EPA), metformin, compound C (AMPK inhibitor), insulin, STO-609 (CaMKK inhibitor), SB203580 (p38 MAPK inhibitor), fluo-3, AM and a monoclonal anti-β-actin antibody were purchased from Sigma Chemical Company (St. Louis, MO, USA). BAPTA-AM (intracellular calcium chelator), monoclonal antibody against ACC and polyclonal antibodies against GLUT4 were purchased from Abcam (Cambridge, MA, USA). Monoclonal antibodies against phosphorylated AMPKα, phosphorylated p38 MAPK, p38 MAPK, phosphorylated AS160, AS160, and the polyclonal antibody against AMPKα were obtained from Cell Signaling Technology (Danvers, MA, USA). The polyclonal antibody against phosphorylated ACC was provided by Merck (Rahway, NJ, USA). Fetal bovine serum (FBS) and penicillin-streptomycin were acquired from Thermo Fisher Scientific (Foster City, CA, USA). The monoclonal antibody against c-Myc was acquired from Santa Cruz Biotechnology (Dallas, TX, USA).

Following steady state, cells were rinsed and incubated in Ca²⁺-free S-MEM supplemented with 5% dialyzed FBS ([Ca²⁺], 5 μM) for 16 h before being switched back to normal medium (α-MEM; [Ca²⁺], 1.8 mM) for the indicated times. Compounds STO-609 (50 μM; Sigma) and dorsomorphin/compound C (50 μM; Sigma) were used as CaMKK and AMPK inhibitors, respectively. Increasing concentrations of dorsomorphin/compound C were tested in order to fully block AMPK activation (Supplementary Figure, panel A, available online at https://doi.org/10.1155/2017/9717353). Alternatively, MDCK cells were exposed to MSC-conditioned medium for 24 h, before exposure to S-MEM for 16 h. The Ca²⁺ switch was then realized using MSC-conditioned α-MEM for the indicated times.

Sulforaphane, the AMPK inhibitor STO-609, the SIRT1 inhibitor EX527, the PGC-1α inhibitor SR-18,292, and LPS were obtained from Sigma (St. Louis, MO, USA). Fluorescein isothiocyanate-dextran (FITC-D4), 2´,7´‐dichlorofluorescein diacetate (DCFH‐DA), and mitoSox red mitochondrial superoxide indicators were obtained from Gibco (Grand Island, NY, USA). Enzyme-linked immunosorbent assays were purchased from Shanghai Enzyme-linked Biotechnology (Shanghai, China) to determine levels of malondialdehyde (MDA), H₂O₂, superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), total antioxidative capacity (T-AOC), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α). CCK-8 kit was purchased from eBioscience (Bender MedSystems, Vienna, Austria). Antibodies against AMPK, phospho-AMPK (p-AMPK), SIRT1, or PGC-1α were obtained from Abcam (Cambridge, UK).

Corticosterone, STO609 (CAMKK inhibitor), A769662 (AMPK activator), glutamate, oligomycin, and carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) are from Sigma-Aldrich S.A.R.L. Doxycycline is from Clontech. Brain-derived neurotrophic factor (BDNF) is from Abnova. Tetramethylrhodamine methyl ester (TMRM) is from Santa Cruz Biotechnology. The antibodies used were as follows: cyto-NR4A1 (ABIN460855, antibodies-online.com); pan-NR4A1 (E6, Santa Cruz Biotechnology); and anti-human NR4A1 (D63C5), phospho (p)-NR4A1 (S350-P), ubiquitin (P4D1), P190A, phospho-acetyl-CoA carboxylase (ACC; S79-P), AMPK and phospho-AMPK (T172-P), HDAC2, and S6 (Cell Signaling Technology). Actin is from Sigma-Aldrich S.A.R.L.; FKBP51 and HSP90 are from BD Biosciences-Europe; GAPDH is from Thermo Fisher Scientific; and GFP and Drebrin (M2F6) are from Abcam. RFP is from Rockland Immunochemicals; PSD-95 is from NeuroMab; synaptophysin is from Thermo Fisher Scientific; and NR1 (ab1516) and NR2 (ab1548) are from Merck Millipore.

The chemical agents were purchased as follows: liraglutide from Novo Nordisk Japan (Chiyoda, Tokyo, Japan); exendin-(9–39) (Ex-9) from AnaSpec (Fremont, CA, USA); l-NAME, SQ22536 (SQ), cAMP-dependent protein kinase (PKA) inhibitor fragment 14–22 myristoylated trifluoroacetate salt (PKI[14–22]), ESI-09, STO-609, and N-omega-nitro-l-arginine methyl ester (l-NAME) from Sigma-Aldrich Japan (Shinagawa, Tokyo, Japan); 10-(4′-[[N-diethylamino]]butyl)-2-chlorophenoxazine (Akt inhibitor X) was purchased from Santa Cruz (Dallas, Texas, USA) and dorsomorphin dihydrochloride from Wako (Osaka, Osaka, Japan).