Polybrene

Short hairpin RNA (shRNA) targeting GTPBP3 was designed and constructed by Hanheng Biotechnology (Shanghai, China). The following sequences were inserted into a lentiviral shRNA vector: forward: 5′-GATCCGACATTGACTTCGGAGAGGATGATAATTCAAGAGATTATCATCCTCTCCGAAGTCAATGTTTTTTTG-3′, reverse: 5′-AATTCAAAAAAACATTGACTTCGGAGAGGATGATAATCTCTTGAATTATCATCCTCTCCGAAGTCAATGTCG-3′. A negative control shRNA directed to the lentiviral vector was generated using 5′-GATCCGTTCTCCGAACGTGTCACGTAATTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTC-3′ (forward) and 5′-AATTGAAAAAATTCTCCGAACGTGTCACGTAATCTCTTGAATTACGTGACACGTTCGGAGAACG-3′ (reverse). R28 cells were cultured until confluence reached approximately 30% and then transfected with lentiviral vectors (titer: 1 × 10⁹) containing GTPBP3 shRNA (KD group) or negative control shRNA (NC group) at a multiplicity of an infection ratio of 20. In brief, 2 × 10⁵ R28 cells were incubated with 40 μL lentivirus, 8 µg polybrene (Hanheng Biotechnology, Shanghai, China) and 1 mL culture medium in a 6-well plate at 37 °C with 5% CO₂ for 4 h. Next, 1 mL fresh growth medium was added per well and the culture medium was replaced with fresh growth medium 24 h later. Puromycin (2 μg/mL) was added to the medium 48 h after transfection to generate stably transfected cells.

Using human PC gene primer sequences, PCshRNA-GFP-puro-lentivirus, scramble-GFP-puro-lentivirus, PC-OE-GFP-puro lentivirus, and vector-GFP-puro were purchased from Hanheng Biotechnology (Hanheng Biotechnology Co., Ltd., Shanghai, China). We previously injected various concentrations of GFP-labeled lentivirus into the cells and measured the effectiveness of the infection, and then applied the obtained optimal multiplicity of infection in subsequent lentivirus infection experiments [44 (link)]. TPC-1 and KTC-1 cells were infected with lentiviruses by plating in six-well plates to reach 60–80% confluence, followed by the addition of 1 mL RPMI-1640 complete culture medium containing 2 mg/mL polybrene (Hanheng Biotechnology Co., Ltd.). Lentiviral supernatant (50 μL) was then collected and added to the dish, followed by 1 mL of culture medium 4 h later. After 48 h, the culture medium was replaced with RPMI-1640 complete medium containing 1 mg/mL puromycin (Sigma-Aldrich, St. Louis, MO, USA) and cultured for 1 week for screening.