Sodium pyruvate

Human malignant melanoma A375 cells (Cat no. SCSP-533) and mouse melanoma B16 cells (Cat no. TCM-2) were ordered from the Cell Bank, Typical Culture Preservation Commission, Chinese Academy of Sciences (Shanghai, China). Cells were cultured with Dulbecco's Modified Eagle's Medium (DMEM)/High glucose (Cat no. SH30243.01B; Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (Cat no. 10091148; Gibco, Invitrogen, Shanghai, China), 1% sodium pyruvate (Cat no. SP0100; Solarbio, Beijing, China), 0.1 U/L penicillin, and 0.1 μg/L streptomycin (Cat no. P1400; Solarbio, Beijing, China). The cells were incubated in 5% CO₂ incubator (HF90, Heal Force Bio-meditech Holdings Limited, Shanghai, China) at 37°C for 48 h and then propagated.

Chitosan (CS), curcumin (CUR), N-isopropylacrylamide, and hyaluronic acid (HA) were purchased from Sigma-Aldrich (Shanghai, China). The 4T1 cells were purchased from the National Collection of Authenticated Cell Cultures (China). Fetal bovine serum (FBS), 0.25% trypsin, 0.02% EDTA, penicillin, streptomycin were purchased from Biological Industries (Beijing, China). The 4T1 cells were grown in RPMI medium supplemented with 5% FBS, 1% penicillin/streptomycin, 1 mM sodium pyruvate (Solarbio, Beijing, China), and 0.4 mM glutamine (Solarbio, Beijing, China) and maintained at 37 °C, 5% CO₂.

Human melanoma A375 cells (Cat no. SCSP-533) and mouse melanoma B16 cells (Cat no. TCM 2) were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences China (Shanghai, China). The cells were cultured in a sterile cell culture chamber (HF240, HEALFORCE, Shanghai Lishen Scientific Equipment Co. Ltd.) with 95% air and 5% CO₂ saturated humidity at a temperature of 37 °C. Human melanoma A375 cells were subsequently cultured and tested using DMEM high glucose medium (Cat no. SH30022.01, Hyclone) supplemented with 10% FBS (Cat no. 10091-148, Gibco), 1% streptomycin mixture (Cat no. P1400, Solarbio), and 1% sodium pyruvate (Cat no. SP0100, Solarbio). Mouse B16 cells were cultured in 1640 medium (Cat no. SH30809.01, Hyclone) supplemented with 10% FBS and 1% streptomycin mixture.

Benzaldehyde (BEN > 99%) was purchased from Chroma Biotechnology Co., Md (Chengdu, China). Parahydroxybenzoic acid (>98%), benzaldehyde (BEN > 99%), acyclovir (ACV > 99%), carbamazepine (CBZ > 99%), vinblastine (VIN > 99%), hydrochlorothiazide (HTZ >99%), and propranolol hydrochloride (PRO >99%) were purchased from Chengdu Alfa Biotechnology Co., Ltd. (Chengdu, China). TMA-DPH was purchased from MedChemExpress (MA, United States). Acetonitrile and formic acid (HPLC grade) were bought from Fisher Chemical (MA, United States). HBSS and MEM were bought from Hyclone (MA, United States), nonessential amino acids solution was purchased from Sigma-Aldrich (Shanghai, China), and sodium pyruvate was bought from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). FBS was purchased from Every Green Biotechnology Co., Ltd. (Zhejiang, China). 0.25% Trypsin–EDTA and penicillin–streptomycin solution was provided by Gibco (NY, United States). DMSO was obtained from Meilunbio (DaLian, China). A 12-well Transwell^® plate was purchased from Corning (NY, United States). All other solvents were of analytical agents and aqueous solutions were prepared by double-distilled water.

The culture and transfection of spermatogonia from the fat greenling were conducted following standardized protocols. The spermatogonia were cultured in L-15 medium supplemented with 10 ng/mL bovine insulin (Solarbio, Beijing, China), 1nM sodium pyruvate (Solarbio, Beijing, China), 1% fish serum, 10 ng/mL glial cell line-derived neurotrophic factor (GDNF, ABclonal, Wuhan, China), 10 ng/mL basic fibroblast growth factor (bFGF, ABclonal, Wuhan, China), and 5% fetal bovine serum (FBS).
For the transfection procedure, cells at the optimal confluence were transfected with prf1-pEGFP-N1 and gzmb-pEGFP-N1 plasmids in the experimental group, and with the empty pEGFP-N1 plasmid in the control group, using Lipofectamine 3000 Transfection Reagent (Catalog No. L3000015, Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Post-transfection, cells were maintained under standard culture conditions and monitored for expression of the transgene. Total RNA was extracted from spermatogonia of both the experimental and control groups, and cDNA was synthesized. The expression of prf1, gzmb, and apoptosis-related genes was analyzed by qRT-PCR (following the procedure described in Section 4.4). The primer sequences are listed in Table S1.

EDM (EDM >99%) and DEDM (DEDM >99%) were purchased from Chengdu Alfa Biotechnology Co., Ltd. (Chengdu, China). Acetonitrile (HPLC grade) was got from fisher chemical (Massachusetts, United States). Non-essential amino acids solution from Sigma-Aldrich (Shanghai, China) and Hank’s balanced salt solution (HBSS), penicillin-streptomycin solution (×100) and MEM were purchased from Hyclone (Massachusetts, United States). Sodium pyruvate was bought from solarbio science and technology Co., Ltd., (Beijing, China). Gibco (New York State, United States) supplied the 0.25% Trypsin-EDTA and penicillin-streptomycin solution. From Every Green Biotechnology Co., Ltd., (Zhejiang, China), FBS was bought. DMSO was obtained from Meilunbio (Dalian, China). 12-well Transwell^® plate was acquired from Corning (New York State, United States). The preparation of aqueous solutions uses double-distilled water and all additional solvents are analytical agents.