Ly294002

L6 rat myogenic cells, subclone C5 (L6-C5) [3 (link),19 (link)], were routinely seeded at the density of 12,000 cells/cm² in DMEM (EuroClone, Milan, Italy) supplemented with 10% Fetal Bovine Serum (FBS) (PAN Biotech, Aidenbach, Germany), 100 units/mL penicillin/streptomycin (Carlo Erba, Milan, Italy) and 10 mM Hepes (PAN Biotech, Aidenbach, Germany). Twenty-four hours after plating, cultures were shifted to a serum-free medium consisting of DMEM supplemented with 1% fatty acid-free Bovine Serum Albumin (Merk, Darmstadt, Germany) [19 (link)]. The cells were treated with 0.1 μM AVP (Sigma, St. Louis, MO, USA) and/or different concentration of LY294002 (Sigma, St. Louis, MO, USA) for 48 h. When AVP and LY294002 were used in combination, LY294002 was added 30 min before the beginning of AVP treatment. Cell cultures were maintained at 37 °C in a 5% CO₂ humidified atmosphere.

Rats were randomly divided into six groups: control group (n = 10), low-dose sevoflurane group (L-Sev; n = 10), high-dose sevoflurane group (H-Sev; n = 10), vehicle group (n = 10), DEX group (n = 10) and DEX + LY294002 (a specific inhibitor of PI3K) group (n = 10). All rats were placed in an anesthesia induction chamber and received anesthesia using an inhalation machine. Oxygen concentration and anesthesia doses were continuously monitored. The control group was treated with 60% O₂ for 2 h, the L-Sev group with 1.5% sevoflurane inhalation for 2 h and the remaining groups (H-Sev group, vehicle group, DEX group and DEX + LY294002 group) were treated with 3% sevoflurane inhalation for 2 h [17 (link)]. One hour prior to sevoflurane treatment, the vehicle group received an intraperitoneal injection of saline, the DEX group received an intraperitoneal injection of 4 μg/kg DEX [18 (link)] and the DEX + LY294002 group received an intraperitoneal injection of 4 μg/kg DEX and intracerebroventricular injection of 25 μg/5 μl LY294002 (Sigma-Aldrich Chemical Company, USA) [19 (link)]. The time at which anesthesia commenced was when sevoflurane concentration had reached a maximum for each group. Gas flow in the anesthesia chamber was maintained at a rate of 4 L/min.

Different final concentrations of H₂O₂ (0, 50, 100, and 200 μmol/L) (Aladdin), characterized by oxidative damage induction^{15 (link)}, were used to induce MSCs apoptosis for the indicated periods (0, 1, 2, and 4 h), and a final concentration of 100 μmol/L for 4 h was used to investigate the protective effect of adropin against MSCs damage (Supplementary Fig. 1). Except those of the control group (Control) cultured under normal condition, other MSCs underwent H₂O₂ treatment and were divided into six groups: (1) the apoptosis model group (H₂O₂): treated with 100 μmol/L H₂O₂ for 4 h; (2)–(4) the low-, moderate-, and high-dose adropin groups (Ad-L, Ad-M, and Ad-H, respectively): treated like the apoptosis model group, but pretreated with 10, 25, and 50 ng/ml adropin (Phoenix Pharmaceuticals, USA) for 24 h, respectively; (5) the LY294002 + Ad-M group (LY): treated like the Ad-M but pretreated with PI3K specific inhibitor LY294002 (Sigma, USA) at 50 μmol/L for 2 h; (6) the PD98059 + Ad-M group (PD): treated like the Ad-M but pretreated with ERK1/2 specific inhibitor PD98059 at 50 μmol/L for 30 min. Cell viability (survival) was assessed by Cell Counting Kit-8 (CCK-8), apoptosis rate by flow cytometry, and the protein expression of Akt, ERK1/2, and antiapoptotic proteins [B-cell lymphoma-2 (BCL-2) and B-cell lymphoma extra-large (BCL-XL)] by western blotting.

Recombinant human or mouse IL-17A was purchased from R&D Systems. The D series resolvins, RvD1 and RvD2, and MK2206 (Akt inhibitor) were from Cayman Chemical. SB202190 (p38 MAP kinase inhibitor), SB216763 (GSK-3β inhibitor), SN50 (NF-κB inhibitor), SP600125 (JNK inhibitor) were obtained from EMD-Millipore. LY294002 (PI3K inhibitor) and LY30351 (inactive analogue of LY294002) were purchased from Sigma-Aldrich. Rabbit polyclonal antibody to Del-1 was from ProteinTech. Rabbit monoclonal antibody (mAb) to β-actin (13E5), rabbit IgG antibodies to total Akt (phosphorylation state independent), phospho-Akt (Ser473), total GSK-3β and phospho-GSK-3β (Ser9), as well as rabbit anti-phospho-C/EBPβ (Thr-188) and anti-phospho-threonine IgG antibodies were purchased from Cell Signaling Technology. Rabbit IgG antibodies to GPR32 and to ALX/FPR2 were from Abcam and Abnova, respectively. FITC- or PE-conjugated goat anti-rabbit IgG and rabbit IgG antibody to C/EBPβ were purchased from Santa Cruz Biotechnology. All reagents (including those mentioned in the description of assays below) were used at doses based on our previously published work or on experiments in this study (see Supplementary Figs 1 and 4).