Polybrene 40804es76

LT-SV40 was subcloned into CV301 vector (Genechem (Shanghai, China)) with blasticidin resistance gene and hTERT was subcloned into CV237 vector (Genechem (Shanghai, China)) with hygromycin resistance gene. Lentivirus was developed by co-transfection into 293T with pHelper 1.0 (Genechem), pHelper 2.0 (Genechem (Shanghai, China)), and the above two vectors. Then, the concentrated viral medium was added into a cell culture medium with 10 µg/ml polybrene (40804ES76, Yeasen). Twenty-four hours later, the culture medium was replaced with the modified EpiCM medium in the presence of blasticidin and hygromycin

The full-length and truncated sequences of TAZ and USP14 were generated by PCR from human cellular cDNA and cloned into the pHAGE-Flag or pHAGE-HA vector. The TAZ^S89A and USP14^C114A plasmids were constructed by site-directed mutagenesis using the inverse PCR from the TAZ-Flag and USP14-HA plasmids. The sequences of gene-specific small hairpin RNAs were designed and synthesized by Sangon Biotech and inserted into the pLKO.1 vector. The primer sequence information was listed in Table S7. Transient transfection was carried out by using polyethyleneimine (25 kDa, Polyscience, US) according to the manufacturer’s protocol. For the generation of stably transduced cell lines, HEK293T cells were cotransfected with the target gene plasmid, the pMD2.G envelope plasmid and the psPAX packaging plasmid. After 72 h, the supernatant was collected and filtered through a 0.45 µm filter (Biofly, Wuhan, China). SW1990 and PANC-1 cells were transduced by treatment with 1 ml of the viral supernatant in the presence of 1 μl of polybrene (40804ES76, Yeasen, Shanghai, China) for 8 h. Transduced cells were successively screened with puromycin dihydrochloride (1 μg/ml) (ST551, Beyotime, China) for 2 weeks. Next, immunoblotting and RT-qPCR were performed to determine the knockdown or overexpression efficiency of the indicated gene.

LSH, USP11, and CYP24A1 knockout cell lines (LSH-KO, USP11-KO, CYP24A1-KO), knockdown cell lines (shLSH, shUSP11, shCYP24A1), and stably overexpressing cell lines were generated using the psPAX2 and pMD2.G lentiviral packaging systems. Briefly, a certain proportion of lentiviral plasmids were transferred into HEK293T cells, after 72 h, the viral supernatant was harvested to infect target cells at 50% confluence with 5 μg/ml polybrene (40804ES76, Yeasen). After 48 h, puromycin was added to screen positive cell pools or clones.