High glucose dmem

Maleimide-functionalized PEG (mPEG-Mal) (Mw ≈ 2 kDa) and t-Boc-NH-PEG-Mal (Mw ≈ 2 kDa) were purchased from JenKem Technology Co. Ltd (Beijing, China). Na₃C₆H₅O₇·2H₂O, CuCl₂, Na₂S, cysteamine, 4,4′-diamino-2,2′-bipyridine (DABPY), tetra-n-butylammonium bromide, furfurylamine were purchased from Energy Chemical (Shanghai, China). MnBr(CO)₅, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydro (EDC), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), myoglobin, and folic acid (FA) were purchased from Sigma-Aldrich (St. Louis, MO, United States). All reagents used were of analytical grade. The water used in all experiments was deionized (DI) water with a resistivity of 18.2 MΩ cm. The molecular weight cutoff of all dialysis bags used in the dialysis process was of 5 kDa. High-glucose DMEM containing 1% penicillin/streptomycin, phosphate-buffered saline (PBS), and trypsin was obtained from KeyGen BioTech Co. Ltd. (Jiangsu, China). Fetal bovine serum was purchased from Absin Bioscience Inc. (Shanghai, China).

L02, Hep3B, Huh7 and HepG2 cell lines were cultured in a 37° C incubator with 5% CO2 and 95% relative humidity for cell line screening via Western blot analysis. The culture medium was made up of high glucose DMEM (KeyGen Biotech, China) supplemented with 10% fetal bovine serum (FBS) (Excell Bio, Shanghai, China). The HepG2 cells were transfected with ALT1-specific siRNA, EP-CAM-specific siRNA, Ki67-specific siRNA, ASPP2-specific siRNA, or negative control siRNA using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.). RNA and protein were extracted at 24 h and 48 h, respectively.

A2780 human OC cells were cultured in high glucose DMEM (KeyGEN, China) supplemented with 10% foetal bovine serum (Gibco, USA) at 37 °C containing 5% CO₂. Three siRNAs targeting the B4GALT5 mRNA region and Nc control siRNA were used for B4GALT5 silencing via transient transfection (RIBOBIO, China). The sequences of siRNAs against B4GALT5 were as follows: GTGGAACAATTTCGGAAAA (si-1); GATCGCAACTATTATGGAT (si-2); CAACCAAATTGGATAAGTA (si-3). Briefly, cells were seeded in a six-well plate 24 h before transfection. When cells reached 60%-70% confluence, Lipofectamine 3000 Transfection Reagent (Invitrogen, USA) was utilized to transfect with siRNAs following the manufacturer’s instructions. For validation, 48 h after transfection, total RNA and proteins were extracted for RT‒qPCR and WB assays as described above.

Methoxy-terminated polyethylene glycol (mPEG, Mw ≈ 2 KDa), carboxylic acid functionalized methoxyl polyethylene glycol (mPEG-COOH, Mw ≈ 2 KDa) were purchased from Ruixi Biological Technology Co., Ltd,. (Xi’an, China). N,N′-carbonyldiimidazole (CDI), 2,2-dithiodiethanol (DIT), succinic anhydride, dextran (Mw ≈ 2 KDa), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-Hydroxy succinimide (NHS), 2-methoxy propylene, pyridine p-toluene sulfonate, propionic anhydride, and dimethylaminopyridine (DMAP) were purchased from Energy Chemical (Shanghai, China). Water used in all the experiments was deionized (DI) water depurated by a Millipore ultrapure water system (Billerica, MA, United States) with a resistivity of 18.2 MΩ cm. High-glucose DMEM containing 1% penicillin/streptomycin, phosphate-buffered saline (PBS), and trypsin were obtained from KeyGen BioTech Co., Ltd,. (Jiangsu, China). Fetal bovine serum (FBS) was purchased from Absin Bioscience Inc (Shanghai, China).