Prime transfection reagent

C57BL/6J mice were purchased from HFK Bioscience (Beijing, China). Mice aged 6–8 weeks were used in experiments and were housed in and maintained under specific pathogen-free (SPF) conditions. The Committee on the Ethics of Animal Experiments of Shandong University approved all the animal studies.
The Hepa1-6 and BNL.CL.2 cell lines were purchased from ATCC (Rockville, USA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO₂ incubator. To construct the pSBbi-luc plasmid, the firefly luciferase fragment was amplified from the pGL6-TA plasmid (Beyotime Biotechnology, China) by PCR, prior to being inserted into the pSBbi-GP plasmid (Addgene, USA) via the Sfil restriction site. Hepa1-6 cells were seeded in 6-well plates overnight, then transfected with 1 μg pSBbi-luc plasmid and 1 μg Sleeping beauty SB100X transposase (BioVector NTCC Inc., China) using the in vitro-jet PRIME transfection reagent (Polyplus Transfection Inc., USA) according to the manufacturer’s instructions. After 24 hours, puromycin (1 μg/mL) was added to screen for stable transfection. The Hepa1-6 cells transfected with these plasmids were named Hepa1-6-GFP-luc⁺ cells.

DNA templates were purchased from RiboBio Company (Guangzhou, China). A detailed list of all DNA templates is provided in Supplementary Table S1. In vitro transcribed siRNAs were synthesized using the In Vitro Transcription T7 Kit (Takara, Japan). A list of all siRNA sequences is provided in Supplementary Table S2.
Hepa1-6 cells were transfected with the relevant vector using the in vitro-jet PRIME transfection reagent (Polyplus Transfection Inc., USA) according to the manufacturer’s instructions. After 48 or 72 hours, the RNA or protein was extracted for subsequent experiments, respectively. DNA-oligonucleotide templates and chemically synthesized siRNA sequences are provided in Supplementary Table S1 and Table S2.