Pgc 1α

The total cell protein was extracted using a radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). The cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were transferred to polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA, USA). The PVDF membranes were blocked with 5% skim milk and were incubated with primary antibodies against PGC-1α (Santa Cruz Biotechnology, Dallas, TX, USA), phospho-protein kinase R-like endoplasmic reticulum kinase (p-PERK; Santa Cruz Biotechnology), PERK, phospho-inositol-requiring enzyme 1α (p-IRE-1α; Santa Cruz Biotechnology), IRE1α, B-cell lymphoma 2 (BCL2; Santa Cruz Biotechnology), Bcl-2-associated X protein (BAX; Santa Cruz Biotechnology), cleaved caspase-3 (Santa Cruz Biotechnology), cleaved poly [ADP-ribose] polymerase 1 (PARP1; Santa Cruz Biotechnology), activating transcription factor 4 (ATF4; Novus Biologicals, Littleton, CO, USA), CCAAT-enhancer-binding protein homologous protein (CHOP; Novus Biologicals), and β-actin (Santa Cruz Biotechnology). After incubation, the membranes were incubated with peroxidase-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (Thermo Fisher Scientific). The protein bands were visualized by using enhanced chemiluminescence reagents (Amersham Biosciences, Uppsala, Sweden).

RNA isolation, cDNA synthesis, and quantitative PCR with the indicated primers (Supplementary Table S3) as well as Western blotting were performed as described^{28 (link)}. The following antibodies were used: NS3, ab13830, CORE, ab2740, HDAC9, and ab18970 (Abcam); FoxO1, 9454, PEPCK, 12940, GAPDH, 2118, ATF2, 9226, Acetylated-Lysine, 9681, PGC-1α, and 2178 (Cell Signalling Technology); and Acetyl-FoxO1, sc49437, PGC-1α, and sc-5815 (Santa Cruz Biotechnology).

DMEM, fetal bovine serum (FBS), bovine serum (BS), and trypsin-EDTA were purchased from ThermoFisher Scientific (United States). Penicillin (10000 units/ml)- streptomycin (10 mg/ml) solution was purchased from Biological Industries (Israel). Nigericin, benzoylbenzoyl-ATP (BzATP), and A438079 were purchased from Sigma-Aldrich (United States). Primary antibodies against β-actin, P2X7, PPARγ, C/EBPα, PRDM16, UCP1, PGC-1α, and horseradish peroxidase-conjugated anti-goat IgG secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (United States). Primary antibodies against SIRT3, SIRT5, phospho-insulin receptor (IR), IR, phospho-insulin receptor substrate 1 (IRS-1), IRS-1, phospho-Akt, Akt, and peroxidase-conjugated anti-rabbit/mouse IgG secondary antibody were purchased from Cell Signaling Technology, Inc (United States).

All common chemicals, including methylthiazol-2-yl-3 (4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), acridine orange (AO), propidium iodine (PI), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and JC-1, were purchased from Sigma (München, Germany). Antibodies against caspase 3, poly(ADP-ribose) polymerase (PARP), PDX-1, MST1, catalase, Nrf2, and PGC-1α were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); β-actin was from Novus Biotechnology (Littleton, CO, USA); RANK was from Cell Signaling Technology (Danvers, MA, USA); pSer536-p65 was from Abcam (Cambridge, MA, USA); and SOD1, SOD2, and Sirt1 antibodies were from GeneTex (Irvine, CA, USA). Denosumab was purchased from GlaxoSmithKline (London, UK). Lentiviruses carrying MST1, RANK, and Sirt1 specific short hairpin (sh)RNAs were purchased from the Taiwan National RNAi Core Facility. All chemicals were dissolved in phosphate-buffered salt (PBS) solution and stored at 20 °C until use in experiments.

Proteins from all the skeletal muscles and cells were extracted using the Protein Extraction Reagent (KeyGENBioTECH, Nanjing, China), and the concentrations were determined with the BCA Assay Kit (KeyGENBioTECH, Nanjing, China). Next, equal amounts of protein were separated on 10% SDS-PAGE gels followed by electrotransfer to polyvinylidenedifluoride membranes (PVDF). After blocking with 5% skim milk powder in 0.05% TBST, the membranes were incubated with the following primary antibodies: FTO, phospho-AMPKα2 (Abcam), AMPKα2 (GeneTex), METTL3 (Proteintech), METTL14 (Sigma), FAS, C/ebpα, ATGL, PGC1α (Santa Cruz), GAPDH (Boster) and β-actin (Hua An, Hangzhou, China). The secondary antibodies were HRP-conjugated anti-mouse and anti-rabbitIgG (Hua An, Hangzhou, China). Finally, the immunocomplexes were detected using the ECL Western Blotting Detection System (Amersham, Biosciences, Piscataway, NJ, USA).

ABA (A0792) was purchased from Tokyo Chemical Industry, ThS (T1892), anhydrous dimethyl sulfoxide (DMSO), triton X-100, paraformaldehyde (PFA), phosphate buffer (PB), phosphate-buffered saline (PBS, pH 7.4), and tris-buffered saline (TBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RIPA buffer (89901) and protease/phosphatase inhibitor cocktail (78445) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Fluorescence mounting medium (S3023) was purchased from Dako (Santa Clara, CA, USA). Primary antibodies against Aβ (6E10; 803001, BioLegend, San Diego, CA, USA), PGC-1α (sc-518025, Santa Cruz Biotechnology, Dallas, TX, USA), PS1 (5643s, Cell Signaling Technology, Danvers, MA, USA), PPAR-γ (2443s, Cell Signaling Technology), p-CREB (9198s, Cell Signaling Technology), CREB (9197s, Cell Signaling Technology), IDE (ab32216, Abcam, Cambridge, MA, USA), LANCL2 (MBS154355, MyBioSource, San Diego, CA, USA), GFAP (Z0334, Dako), Iba-1 (019-19741, Wako Chemical, Osaka, Japan), and NEP (AF1126, R&D Systems, Minneapolis, MN, USA) were used. β-actin (sc-47778HRP, Santa Cruz Biotechnology) and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Alexa Fluor secondary antibodies were purchased from Thermo Fisher Scientific.