Hitransg

To knock down each candidate gene in 231-BR cells, the lentiviral vector (U6-MCS-Ubiquitin-Cherry-IRES-puromycin) containing the short-hairpin RNA (shRNA) specifically targeting each gene was constructed (GeneChem, China). For lentivirus infection, three individual shRNA oligos targeting each gene were pooled together: see Supplementary Table 3, and the HitransG (Genechem) was used according to the manufacturer’s instructions. Student’s t-test was used to test for significance. P values of < 0.05 were defined as significant.

Lenti-viruses expressing THBS1, THBS1-shRNA (target sequences: gc GCGTGTTTGACATCTTTGA), scrambled shRNA, and vector purchased from Genechem (Shanghai, China). In brief, 3 × 10⁴ cells were seeded into a 6-well plate and then transfected with the specified lenti-virus using HitransG (Genechem, Shanghai, China). In addition, infected cells were selected by 2.5 μg/ml puromycin (MCE, USA) for > 14 days, and verified by RT-qPCR.

ANXA2 and TTK small interfering RNAs (siRNAs) were purchased from BioeGene (Shanghai, China), FLAG-ANXA2 and MYC-TTK plasmids were purchased from Genomeditech (Shanghai, China), and lentiviral vectors for ANXA2 knockdown were purchased from GeneChem (Shanghai, China). TSnanofect (Tsingke, China) was used to transiently transfect siRNAs and plasmids into esophageal cancer cells and HeLa cells. Esophageal cancer cells were infected with lentivirus by HitransG (GeneChem) to establish stably transfected cell lines. Cells screened for further experiments were continuously cultured at a concentration of 0.5 μg/mL puromycin. The siRNA and shRNA sequences used in this study are listed in Supplementary Table 1.

Lentiviral transduction was utilized to overexpress miR-100 in MCL cell lines (Jeko-1, Mino). The lentiviruses contained human miR-100-up-GFP (LV-miR-up), the control (LV-NC-up), LV-mTOR-RNAi-GFP (LV-mTOR-RNAi) and the control (LV-NC-RNAi). The plasmids containing the 3′-UTR of the mTOR luciferase plasmid, miR-100 plasmid, Renilla plasmid, and mTOR-up-GFP plasmid were purchased from Genechem Company (Genechem Co, Ltd, Shanghai, China). The MOI of Jeko-1 was 40, and the MOI of Mino was 60. The lentiviruses were added to 2 × 10⁵/mL cells in 6-well plates with 1 ml of RPMI‑1640 containing no FBS and 40 μl/mL HitransG (Genechem Co, Ltd, Shanghai, China). Fresh culture medium contained 15% FBS after 16 h. Cells were harvested after 72 h of transduction, and qRT‑PCR was performed to confirm the efficiency of transduction.

HCC cell lines HepG2 and Huh-7 cells were purchased from Shanghai cell bank of the Chinese Academy of Science. The normal human hepatocytes LO2 cells were provided by Wuhan University Medical Science Research Center. These three types of adherent cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO₂. Cells were grown to 75% to 80% confluency and collected by trypsinization with 0.25% trypsin-EDTA.
For lentivirus infection, the HitransG (Genechem, Shanghai, China) was used according to the manufacturer’s instructions. HepG2 cells were infected at a high multiplicity of infection (MOI = 20) with LV-WDR45B-RNAi and blank lentivirus. When lentiviral infection efficacy reached around 80%, 2.0 μg/mL puromycin was used to screen the stable cells. A qRT-PCR was performed to verify the efficiency of the WDR45B knockdown.

The shSIRT5 lentivirus was packaged by GeneChem Co. (Shanghai, China) using U6-MCS-Ubiquitin-mCherry-puromycin vector and added to Reh-6-MPR and Nalm-6-6-MPR cells at a multiplicity of infection (MOI) of 60 with HiTrans G (GeneChem Co.). Positively infected cells were selected with puromycin (1 μg/mL; ST551, Beyotime, Shanghai, China).

For lentivirus construction, oligonucleotides with targeting sequences were used for the cloning of small-interfering RNA (siRNA) in the hU6-MCS-CMV-puro lentiviral vectors (GeneChem Co., Shanghai, China). The three recombinant lentiviruses with siRNA-SCD1 were produced by co-transfection of 293T cells with plasmids pHelper 1.0 and pHelper 2.0 (GeneChem Co.). Lentivirus-containing supernatant was harvested 48 h after transfection and concentrated by ultracentrifugation (2 h at 50,000 × g). The detailed sequences of target siRNA are listed in Supplementary Table S4. Transfection of the siRNAs was performed with Hitrans G (GeneChem) according to the manufacturer’s instruction. Stably transfecting clones were validated by quantitative real-time RT-PCR (qRT-PCR).
Overexpression plasmids and siRNAs of YAP were transfected into cells using Lipofectamine 2000 (Invitrogen). The knockdown sequence of YAP was as follows: siYAP, 5′-GACATCTTCTGGTCAGAGA-3′. A full-length YAP cDNA was synthesised by GeneChem Company (Shanghai, China), and cloned into plasmid-CMV/MCS//SV40/Neomycin. At 48 h post transfection, the modified cells were harvested for western blot and qRT-PCR validation.