Blank plvx puro

Human high metastatic HCC cell line MHCC-97H and low metastatic HCC cell line MHCC-97L, and immortalized normal human liver cell line LO2 were obtained from ATCC (Manassas, VA, USA) and cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, Logan, UT, USA) complemented with 10% fetal bovine serum (FBS) (Gibco, Detroit, MI, USA) along with 1% Penicillin-Streptomycin solution separately incubated in 37 °C with 5% CO₂ and 95% air.
Oligonucleotides encoding short hairpin RNA (shRNA) targeting human TRIM52 (point 670–692, 5’-GGGCATGTGCTTTAAACAC-3′) and scramble shRNA were cloned into the pLKO.1 lentiviral vector. The cDNA encoding TRIM52 and PPM1A was obtained by reverse transcription PCR (RT-PCR) and cloned into pLVX-Puro for constructing pLVX-Puro-TRIM52 and pLVX-Puro-PPM1A expressing vector, respectively. pLKO.1-scramble shRNA (NC) and blank pLVX-Puro (Vector) were used as the negative controls. 293T cells were plated in 6-well plates and transfected with constructs for 4–6 h using Lipofectamine Reagent (Invitrogen, Carlsbad, CA, USA) according to the instructions of the manufacturer. After incubation in a CO₂ incubator at 37 °C, recombined lentivirus was collected 48 h after transfection and used for MHCC-97H and MHCC-97L cells infection.

H9C2 cardiomyocytes were seeded in the six-well plate for 24 h and that with 50 nM miR-3568 mimic (sequence: 5′-UGUUCUUCCCGUGCAGAAGCAG-3′), miR-3568 inhibitor (sequence: 5′-CUGCUUCUGCACGGGAAGAACA-3′), or relative negative controls (NC) transfection for 6 h were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the instruction of the manufacturer. TRIM62 ectopic expression lentiviral vector was constructed by integrating the coding sequence (CDS) of TRIM62 into pLVX-Puro. The primers used to synthesize the CDS were as follows: forward: 5′-GCGAATTCATGGAGGAGAACAATGACT-3′; reverse: 5′-CGGGATCCCTGAATCCATATTGTGTTT-3′. 293T cells were seeded in the six-well plate and that with pLVX-Puro-TRIM62 or blank pLVX-Puro transfection for 4–6 h were performed using Lipofectamine reagent according to the manufacturer’s protocol. Viruses were collected after 48 h transfection and were used to infect H9C2 cardiomyocytes. The blank pLVX-Puro (Vector) was used as NC.