Penicillin streptomycin mixture

Normal human astro glia cell HEB cell line and human glioma cell lines U251 and SHG44 and human glioblastoma U-87 MG cell line (Chinese Type Culture Collection, Shanghai, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Scientific HyClone, Beijing, China) supplemented with 50 U/ml of a penicillin/streptomycin mixture (Solarbio Biotech Corp. Beijing, China) and 10% fetal bovine serum (Sijiqing Biotech Corp, Hangzhou, China). All cells were routinely grown in 75-cm² cell culture plates (Corning Inc., Corning, NY, USA) at 37°C with 5% CO₂ in a humidified atmosphere. The cells were collected in logarithmic phase for the following experiments. On the day before transfection, cells were digested by trypsin (0.25%, Solarbio Biotech Corp., Beijing, China), counted and seeded in a six-well plate at an optimal concentration. When the cells achieved 80% confluence, the medium was changed with serum-free DMEM, and cells were incubated overnight. Control siRNA or siRNA targeting CHL1 (10 nM for both; Table 1) complexed with Entranster™-R4000 (Cat. No. 4000-3, Engreen, Beijing, China) was transfected into three cell lines. In the vehicle control group, cells were treated with the same volume of transfection reagent. The efficiency of CHL1 siRNA was confirmed by RT-PCR and Western blot.

Zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Terephthalic acid (H₂BDC) and dimethyl sulfoxide (DMSO) were obtained from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Triethylamine (TEA) was acquired from Tianjin Fuchen Chemical Reagent Factory (Tianjin, China). Trichloromethane (CHCl₃) and N,N-Dimethylformamide (DMF) were got from Beijing Chemical Factory (Beijing, China). ORI was purchased from Nantong Feiyu Biotechnology Co., Ltd. (Nantong, China). Methanol (Chromatographic reagent grade) was obtained from Thermo Fisher Scientific (Shanghai, China). PBS and penicillin-streptomycin mixture were purchased from Solarbio (Beijing, China). High-glucose Dulbecco’s Modified Eagle Medium (DMEM) was obtained from Corning (Manassas, VA, USA). Fetal bovine serum was available from Zhejiang Tianhang Biotechnology Co., Ltd. (Zhejiang, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-ltetrazolium bromide (MTT) was obtained from Beijing Biodee Biotechnology Co., Ltd. (Beijing, China). 4′,6-diamidino-2-phenylindole (DAPI) and Annexin V-FITC Apoptosis Detection Kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). All chemicals were of analytical grade or higher.

Fetal bovine serum and RPMI-1640 medium were purchased from GIBCO, USA; trypsin-EDTA digestion solution, MTT, and DMSO were purchased from Sigma, USA; penicillin streptomycin mixture and OPTI-MEM were purchased from Solarbio, USA; Tris balanced phenol was purchased from Thermos, USA; 60 mm/100 mm Petri dishes and 6/12/24/48/96-well cell culture plates were purchased from CORNING, USA; RIPA lysate and BCA protein concentration kits were purchased from Beyotime; antibodies and protease inhibitors were purchased from Proteintech.

Hericium erinaceus was provided by Guohua Agriculture and Forestry Technology Development Co., LTD (Ankang, China). Fluo-3AM, papain (800 U/mg), neutral protease (100 U/mg), Protamex (120 U/mg), pepsin (3,000 U/mg), alkaline protease (200 U/mg), and trypsin (2,500 U/mg) were bought from Shanghai Biotechnology Co. LTD (Shanghai, China). The Caco-2 cell line was provided by the China Center for Type Culture Collection (Wuhan, China). The penicillin–streptomycin mixture, fetal bovine serum (FBS), CCK8 kit, non-essential amino acid (NEAA) solution, and DMEM medium were bought from Solarbio Co., Ltd. (Beijing, China).

The primary NP cells used in this study were isolated from normal or degenerative NP tissues (n = 6) and cultured in Dulbecco's modified Eagle medium supplemented with foetal bovine serum (10%; 16000‐044, GIBCO, USA) and penicillin‐streptomycin mixture (1%, P1400‐100, Solarbio, China) at 37°C with 5% CO₂. The AKT agonist IGF‐1 (20 ng/mL; 100‐11, Peprotech, USA), PI3K inhibitor LY294002 (10 μM, S1105, Selleck, USA) and MG132 (10 μmol/L; Selleck, USA) were dissolved in DMSO (D2650, Sigma, USA) and used for cell culturing. The sequences of the microRNA negative control (NC) and miR‐338‐3p mimic and inhibitor are listed in Supplementary File S1.