Trypsin

For primary PA cells, PA tissues were minced and digested in trypsin (cat: G4001, Servicebio, Wuhan, China) for 30 min and then cultured in DMEM containing 10% fetal bovine serum for subsequent experiments.
For primary fibroblasts, tumors and skin tissues were minced and digested in type I collagenase (cat: 40507ES60, Yeasen, Shanghai, China) and then cultured in DMEM containing 10% fetal bovine serum at 37 °C until fibroblasts attached to the dish. Anti-Fibroblast MicroBeads (cat: no. 130-050-601 and no. 130-116-474, Miltenyi Biotec, Bergisch Gladbach, Germany) and anti-fibroblast-activating protein (cat: ab218164; Abcam, Cambridge, UK) were prepared to isolate TAFs and NFs. The cells were incubated with FAP antibodies in ice for 1 h. After washing, microbead-coupled secondary antibodies were added, and FAP+ cells were separated into magnetic columns after rinsing. Primary fibroblasts were used prior to passage 5.
Primary fibroblasts of rats and mice were derived from skin tissues, and the extraction method was consistent with that of human primary fibroblasts. For primary TAF cells of rats and mice, tumor cells were mixed with primary fibroblasts for tumor grafting in the ratio of 3:1. Tumor tissues were digested after the tumor was removed, and the extraction method was consistent with that of human primary fibroblasts.

Human CRC cell line HCT116 and HT29 were bought from China Center for Type Culture Collection (Wuhan, CN). Cell lines were recently authenticated by STR profiling and tested for mycoplasma contamination using TransDetect® PCR Mycoplasma Detection Kit (TransGen Biotech, Peking, CN). Cells were cultured on the rigid dish with McCoy’s 5 A Medium (Hyclone, Utah, USA) containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, USA) and 1% Penicillin-Streptomycin (Hyclone, USA) in 37 °C with 5% CO₂. Cells were passaged with 0.25% Trypsin (Servicebio, Wuhan, CN) every 3–4 days.

Healthy spotted sea bass weighing 28.46–31.35 g were anesthetized with 40 mg mL⁻¹ 3-aminobenzoic acid ethyl ester methane-sulfonate (MS-222) and then wiped with 75% ethanol. The dorsal white skeletal muscle (1 cm³) was immediately taken and washed four times in phosphate-buffered saline (PBS) with 1% 100 U mL⁻¹ penicillin and 100 µg mL⁻¹ streptomycin (Solarbio, Beijing, China). Next, the skeletal muscle tissues were cleaned by removing adipose tissues, cut into small pieces (1 mm³), evenly distributed into a 175 cm² standard cell culture chamber, supplemented with growth medium (GM) (L-15 medium (G-CLONE, Beijing, China) mixed with 20% fetal bovine serum (FBS, ABSIN, Shanghai, China), 10 ng mL⁻¹ bFGF (Solarbio, Beijing, China), 1% 100 U mL⁻¹ penicillin and 100 µg mL⁻¹ streptomycin), and then cultured at 25 °C in a CO₂-free incubator (Jinghong, Shanghai, China). Every 3–4 days, the whole medium was replaced with fresh medium until cell emigration and passaging. When the primary cells grew to approximately 70% abundance, 0.25% trypsin (Servicebio, Wuhan, China) was used for subculture, and the isolated cell suspension was precultured in the incubator for 2 h to remove the fibroblasts.

Nucleus pulposus cells were isolated from different donors and these cells were used for the cell-based in vitro experiments in this study. The isolation process of NPCs was performed as described previously (Zhang et al., 2018 (link)). Nucleus pulposus tissue samples were washed three times with PBS, then cut and digested using 0.25% trypsin (Servicebio, Wuhan, China) and 0.01% EDTA at 37°C for approximately 45 min. Next, tissue pieces were digested by type II collagenase (Servicebio) for 4 h at 37°C. The cell suspension was sieved through a cell strainer (70 μm; Beyotime, Shanghai, China) and centrifuged at 1,000 × g for 5 min. NPCs were resuspended in DMEM/F12 with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Beyotime) and cultured in a humidified atmosphere of 5% CO₂ at 37°C, changing medium every 3 days. Cells at second passage were used for subsequent experiments. To establish the degeneration model of NPCs, cells were incubated for 24 h then treated with Recombinant Human Interleukin 1β (IL-1β) (10 ng/mL, Beyotime) for 24 or 48 h. Total RNA or protein were then extracted (Bai et al., 2019 (link); Zhang et al., 2019 (link)).

DAT was prepared according to previous report^{43 (link)}. Briefly, subcutaneous fat harvested via liposuction from donors at the Wuhan Union hospital and subjected to three freeze–thaw cycles. With washing and centrifuging at 1200×g for 5 min, the samples separated into three layers and the middle matrix layer was collected and then digested with 0.25% trypsin and 0.1% EDTA (Servicebio, China). After enzymatic digestion, the tissues were washed and subjected to polar solvent extraction using isopropanol at for 48 h 37 °C. The samples were subsequently treated with enzymatic digestion solution containing 20 ng/mL DNAse (Sigma, USA), and 20 ng/mL RNAse (Sigma, USA) for 16 h at 37 °C. Then, the processed tissues were rinsed with PBS 3 times and subjected to another 48 h’ extraction using isopropanol to remove reminded lipid. After several washes with PBS, DAT was collected and stored in absolute ethanol at 4 °C until needed.
Adipose tissue and prepared DAT were dehydrated via a graded alcohol series and embedded in paraffin. After cut the samples into sections in 5 μm thickness, the DAPI staining was performed to investigate the nucleus in tissues. Hematoxylin & Eosin (HE), Masson's Trichrome (Masson), and Oil Red O staining were conducted to distinguish the tissue structure changes between adipose tissue and DAT.

UP302 was purchased from Nanjing m&m biotechnology Co. Ltd. The following reagents were used: fetal bovine serum (Procell, China), Dulbecco's Modified Eagle Medium (DMEM), Iscove's Modified Dulbecco's Medium(IMDM), trypsin (Servicebio, China), CCK8 (Sparkjade, China), chloroquine (Sparkjade, China), PI and FITC, Mitochondrial membrane potential assay kit with JC-1 and Reactive Oxygen Species Assay Kit (Beyotime, China), Antibodies against Caspase-3, BAX, BCL-2, LC3B, AKT123, PI3K, P-PI3K, P-AKT, AIF, Tomm20, PINK1, Parkin, Beclin1, and β-actin were purchased from ABclonal(China). Twelve Balb/c female nude mice, aged four weeks, were purchased from Jinan Pengyue Experimental Animal Breeding Co. LTD.