Puromycin

To generate GSK3α/β or PD-L1 knockout cancer cell lines, the respective target sequences were cloned into lentiCRISPRv2 vector. Cells were transiently transfected with the recombinant plasmids using Lipo6000 Transfection Reagent (C0526, Beyotime), followed by puromycin (A610593, Sangon) selection. Survived cells were plated in limiting dilution in 96-well plates to isolate single-cell colonies. Knockout of the target genes was confirmed by western blot.
To establish stable cell lines overexpressing PD-L1-v1/v4, CDK13 or RPL22−3×HA, the coding sequences were cloned into pCDH-CMV-MCS-EF1-Puro vector (CD510B-1, System Biosciences). Packaging of lentivirus was carried out in 293T cells by co-transfecting the recombinant plasmids and virus packaging vectors (pMD2.G and psPAX2). Virus-containing medium was concentrated using 3×PEG collection medium. Cells were infected with virus in the presence of 10 µg/mL polybrene and screened by puromycin.

3T3L1 preadipocytes (ATCC Cat# CL-173, RRID:CVCL_0123) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. The 3T3L1 preadipocyte lines were maintained in 10% calf serum supplemented with puromycin (Sangon, A610593).

The target sequences of Mybbp1a (5′-GCUGGUGAAUGUGCUGAAGAUGGCC-3′),
and IGFBP5 (5′-CTCCCATTCTCCAATGACT-3′) were obtained from Shanghai Genechem Co., Ltd. and used to generate stable Mybbp1a or IGFBP5 knockdown cell lines according to the instructions of manufacturer. Cells were selected by puromycin (4 μg/ml) (Sangon, Shanghai, China) for 72 h, and the transfection efficiency was proved by quantifying EGFP fluorescence, RT-PCR and western blotting. Stably transfected Huh7 and HCC-LM3 cells were maintained in the Modified Eagle medium (MEM, Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS, Gibco) for further study.