Gdf11

PC12 cells were purchased from zqxzbio (Shanghai, China) and cultured with complete medium (DMEM supplemented with 5% (v/v), fetal bovine serum (FBS; Procell Life Science & Technology Co., Ltd., Wuhan, China), 10% (v/v) horse serum (HS; Biological Industries, Israel), 100 U/mL penicillin and 100 µg/mL streptomycin) at 37 °C, and 5% CO₂ in a humidified atmosphere. After reaching approximately 80% confluence, PC12 cells were trypsinized and sub-cultured. For following experiments, the medium was changed to starved medium (DMEM supplemented with 0.5% FBS, 1% HS, 100 U/mL penicillin and 100 µg/mL streptomycin) after cultivation overnight. After 6 h of starvation, different concentrations of GDF11 (12.5, 25, 50 and 100 ng/mL; R & D systems, Minneapolis, MN, USA) were added, respectively. For signal pathway inhibition, after 6 h starvation, PC12 cells were pretreated with ALK5 inhibitor SB431542 (Beyotime Biotechnology, Shanghai, China) or PI3K inhibitor LY294002 (Beyotime Biotechnology, China) for 4 h before GDF11 was added for a further 5–72 h.

Briefly, cells were seeded in 96-well cell culture plate at a density of 1×10 ⁴ cells per well, then incubated for 24 hours. Then the medium was removed and the cells were treated with new FBS free medium followed by the addition of CCK-8 solution (Dojindo Laboratories, Mashikimachi, kamimashiki gun Kumamoto, Japan). After 2 hours, the BioTEK (Winooski, VT) was used to record the absorbance at 450 nm. In some experiments, the cytokines/growth factors were added into the OC culture medium for 24 hours with different doses. The factors are IGF-1 (R&D Systems, Minneapolis, USA, 791-MG-050), GH (ProSpec, Rehovot, Israel, CYT-540), Wnt3a (R&D Systems, Minneapolis, USA, 1324-WN-010), TGF-β1 (R&D Systems, Minneapolis, USA, 7666-MB-005) and GDF11(R&D Systems, Minneapolis, USA, 1958-GD-010).

Blood draws were performed via venipuncture by a trained phlebotomist. Following a 10-h fast, all subjects submitted a blood sample for analysis in the morning to control for diurnal variations. Blood was drawn from the antecubital vein into dry serum tubes (Vacutainer, Becton, Dickinson and Company, Franklin Lakes, NJ). Upon clotting, blood was centrifuged and serum was collected for analysis. Serum was centrifuged at 5000 g for 5 min to pellet debris. Irisin (Biovision, Milpitas, CA); myonectin (Aviscera Bioscience Santa Clara, CA); GDF-11 (San Diego, CA); cortisol (Spring Valley, CA); FGF-21, IL-6, IL-15, and CRP (R & D Systems, Minneapolis, MN) were measured using quantitative sandwich ELISA kits, following the instructions provided for each kit. Final reactions were measured using a spectrophotometer (Molecular Devices, Sunnyvale, CA) at 450 nm optical density and final concentration of the samples was calculated using SoftMax Pro 5.4 (Molecular Devices, Sunnyvale, CA) via standard curves for reference.

Cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China, P0013B). Total protein was quantified by BCA protein assay (Bio-Rad, Berkery). Each sample was adjusted to equal amount of protein using 5× loading buffer for loading. The samples were separated by SDS-PAGE, transferred to a polyvinylidene fluoride membrane and immunoblotted with the following antibodies: GDF11(19581,R&D Systems, Emeryville, CA, USA), VEGF (ab46154, Abcam, USA), HGF (ab83760, Abcam, USA), phospho-AKT (#4060, Ser473, Cell Signaling Technology), AKT (#4685, following Abs are all from Cell Signaling Technology), Phospho-PI3 Kinase (p85 (Tyr458)/p55 (Tyr199), #4228), PI3K (#4257), BCL2 (#2827), BAX (#14796), Cleaved Caspase3 (#9661), anti-β-actin (#3700), Cleaved Caspase3 (#9661), at 4 °C overnight. After incubation of the membranes with peroxidase-conjugated secondary antibodies (Cell Signaling Technology), bands were visualized using enhanced chemiluminescence reagents (Bio-Rad, USA).

Human nodal (Q96S42), BMP7 (P18075), BMP15 (O95972), BMP8b (P34820), and GDF11 (O95390) were obtained from R&D Systems or PeproTech. Activin A (P08476), activin B (Q53T31), and BMP10 (O95393) were produced in-house using stably transfected CHO cells. Activin A was captured from CM by Protein A affinity chromatography. BMP10 was captured from CM by Metal affinity chromatography (Excel, Cytiva). Both GF moieties were separated from the prodomain using Reversed Phase Chromatography (Resource RPC, Cytiva). Activin B was purified as described (50 (link)). GFs were lyophilized and stored at −80 °C. NCBI-protein accession numbers are shown in parentheses.

All antibodies used in this study are summarized in Table 1. The recombinant human GDF-11 and BMP-4 were obtained from R&D systems. The SB431542 was obtained from Sigma. GDF-11 and BMP-4 were solubilized in phosphate-buffered saline (PBS). SB431542 was dissolved in dimethyl sulfoxide (DMSO).Table 1

The information of antibodies

Name of antibody	Manufacturer and catalog #	Molecular weight	Dilution used
CGB	Proteintech (11615–1-AP)	18 kDa	3000x
Phospho-SMAD1/5/8	Cell Signaling Technology (13820)	60 kDa	1000x
Phospho-SMAD2	Cell Signaling Technology (3108)	60 kDa	1000x
Phospho-SMAD3	Cell Signaling Technology (9520)	52 kDa	1000x
SMAD1	Cell Signaling Technology (6944)	60 kDa	1000x
SMAD2	Cell Signaling Technology (3103)	60 kDa	1000x
SMAD3	Cell Signaling Technology (9523)	52 kDa	1000x
SMAD4	Cell Signaling Technology (38454)	70 kDa	1000x
α-Tubulin	Santa Cruz (sc-23948)	55 kDa	5000x