Ara a

Cells were seeded in triplicate at 1 × 10³/ mL in a twofold serial dilution of either GCV (Sigma), hydroxyurea (Sigma), or ara-A (Sigma). The ara-A plate also contained 1 µM EHNA (Sigma) to prevent deoxyadenosine deamination (42 (link)). After 72-h incubation at 37°C, 20 µL resazurin sodium salt (alamarBlue, Sigma) was added at 0.5 mM in PBS to each well and incubated for a further 6 h. The fluorescence signal was determined using an Infinite 200 pro plate reader (Tecan) using an excitation wavelength of 540 nm and an emission wavelength of 590 nm. Data were analyzed, to derive EC₅₀ values and statistics using Prism (GraphPad). RNAi knockdown strains were induced with 1 µg/mL tetracycline 24 h prior to plating.

GYY4137 [morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate], DTT (DL-dithiothreitol), AMPK inhibitors Compound C (6-[4-[2-(1-piperidinyl)eth-oxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine), Ara-A (ATP-mimetic, 9-β-D-arabinofuranoside), and MMTS (S-methyl methane thiosulfonate) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Biotin-HPDP and HRP (horseradish peroxidase)-conjugated streptavidin were purchased from Thermo Scientific (Rockford, IL, United States). Primary antibodies for phospho-AMPKα (Thr172), AMPKα, phospho-CaMKKβ (Ser458/495), and CaMKKβ were bought from Cell Signaling Technology (Boston, MA, United States). Primary antibodies for NeuN (neuron-specific nuclear protein), c-Fos, and NPY were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Primary antibody for GAPDH (glyceraldehyde-3-phosphate dehydrogenase), HRP-conjugated secondary antibodies, fluorescent secondary antibodies, and bovine serum albumin (BSA) were obtained from Beyotime Biotechnology (Shanghai, China). Other agents were all purchased from commercial suppliers. Biotin-HPDP was freshly dissolved in dimethysulfoxide (DMSO) and other drugs were prepared freshly with double-distilled water or buffer solutions to the final concentrations before application.

P-AMPK (SC-33524), AMPK (SC-25792), p-PI3K (SC-129289), p-PI3K (SC-1637), p-ERK (SC-7383), ERK (SC-1647) were purchased from Santa Cruz Biotechnology, Inc. (final dilution of 1:1000 for western blotting, Santa Cruz, CA, USA). Omentin-1 (A7234), IL-4 (A4988), TNF-α (A11534), Cluster of Differentiation 68 (CD68) (GTX41865), CD86 (GTX34569), and CD163 (NBP2-36495) were purchased from ABclonal, Inc., GeneTex, Inc. and Novus Biologicals (final dilution of 1:100 for Immunohistochemistry staining or Immunofluorescence staining). PI3K, AMPK, ERK, IL-4, and control ON-TARGET plus siRNAs were purchased from Dharmacon, Inc. (Lafayette, CO, USA). The qPCR primers and the Taqman ® one-step PCR Master Mix were supplied by Applied Biosystems (Foster City, CA, USA). Pharmacological inhibitors for PI3K (LY294002, 1 μM, catalog number: 440202; Wortmannin, 1 μM, catalog number: W1628), AMPK (Ara A, 1 μM, catalog number: P5499; Compound C, 1 μM, catalog number: 171260), ERK (ERK II, 1 μM, catalog number: 328007) and all other chemicals not previously mentioned were provided by Sigma-Aldrich (St. Louis, MO, USA).