Igf 1

Thyroid tissues were cultured in RPMI 1640 medium (WISENT, Nanjing, China, 350‐000‐CL) supplemented with PBS, spleen homogenate or 1 ng mL⁻¹ IGF‐1 (MedChemExpress, New Jersey, USA, HY‐P7070), and the inhibitor of the IGF‐1 receptor (picropodophyllin, AXL1717) (MEC, HY‐15494) for 6 h. Afterward, the tissues were subjected to Western blot analysis.
For the Western blot, tissue samples were lysed in RIPA buffer (Beyotime, P0013B) containing protease inhibitor PMSF (Beyotime, ST2573) at 4 °C for 30 min. The lysates were centrifuged at 12,000 rpm for 10 min, and the protein extracts were collected.
The protein extracts were separated by SDS‐PAGE electrophoresis and transferred to a PVDF membrane using the Mini‐PROTEAN Tetra Electrophoresis System (Bio‐Rad). The PVDF membrane was then incubated overnight at 4 °C with primary antibodies against Cyclin D1 (Abcam, ab134175, 1:10 000) or GAPDH (Proteintech, HRP‐60004, 1:10 000).
To detect the bound antibodies, a horseradish peroxidase (HRP)‐linked anti‐rabbit secondary antibody (Jackson ImmunoResearch, 111‐035‐003) was used. The HRP signal was visualized using 4200SF (Tanon Shanghai). This allowed for the visualization of Cyclin D1 and the internal control protein GAPDH on the PVDF membrane.

Thyroid tissues were cultured in RPMI 1640 medium (WISENT, Nanjing, China, 350‐000‐CL) supplemented with PBS, spleen homogenate or 1 ng mL⁻¹ IGF‐1 (MedChemExpress, New Jersey, USA, HY‐P7070) for 6 h. After the culture period, the tissues were subjected to EdU staining to detect proliferating cells. EdU labeling was performed using the BeyoClick EdU Cell Proliferation Kit with Alexa Fluor 647 (Beyotime, C0081S) following the manufacturer's instructions.

HUVECs (BNCC Co. Ltd.); fetal bovine serum (10%, HyClone); Penicillin–streptomycin solution (1%, HyClone); Optical microscope (DM2700 M; Leica Microsystems Technology Co. Ltd.); confocal microscopy (Olympus); whole cell lysis reagent (KeyGen Biotech); polyvinylidene fluoride (Beyotime); primary antibodies including anti‐Akt (1:1000, Abcam), anti‐p‐Akt (1:1000, Abcam), anti‐β‐actin (1:1000, CST, Boston), anti‐VEGF (1:1000, Abcam), anti‐Pi3k (1:1000, Abcam), anti‐p‐Pi3k (1:1000, Abcam), anti‐Col‐I (1:1000, CST, Boston); a gel and blot imaging system (Syngene); LY294002 (HY‐10108, MedChemExpress); IGF‐1 (HY‐P700478, MedChemExpress).

SCs were plated in 6-well plates (8 × 10⁴/well) for 24 h, after which fresh media with or without 1 nM EpoB was added for an additional 24 h. In some experiments, 3-methyladenine (3-MA; 0.5 mM), IGF-1 (100 ng/ml), or LY294002 (20 μM; MedChemExpress, Shanghai, China) was added to the cultures and then maintained in the same media for the entire experiment. Cells were then suspended in 195 μl Annexin V-FITC binding buffer to which 5 μl Annexin V-FITC and 10 μl PI (Beyotime, Shanghai, China) was added. Following a 20 min staining period, flow cytometry (Beckman Instruments, Pasadena, CA, USA) was used to analyze samples in triplicate.

C2C12 myoblasts were purchased from ATCC (Shanghai, China). Dulbecco modified Eagle’s medium (DMEM), penicillin/streptomycin (P/S), fetal bovine serum (FBS), and horse serum (HS) were purchased from Thermo Scientific (Waltham, MA, USA). IGF-1, BMS754807 (BMS), 3-MA, and chloroquine (CQ) were purchased from MedChemExpress (Shanghai, China). Antibodies such as anti-phospho-IGF-1R, anti-IGF-1R, anti-phospho-AKT, anti-AKT, anti-phospho-mTOR, and anti-mTOR were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-BNIP3, anti-myosin heavy chain (MHC), anti-Myogenin (MyoG), and anti-TOMM20 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies such as anti-α-tubulin, anti-GAPDH, anti-PGC-1α, anti-p62, and anti-LC3 were obtained from Protientech (Wuhan, China).