Polybrene

Lentivirus to overexpress rat and human NFIC and control plasmid DNA were designed and constructed (Hanbio, Shanghai, China). Lentivirus was utilized to transfect SCAPs (MOI = 35) and 293T cell line (MOI = 20) at 50%–60% confluence with the incubation of 4 μg/mL Polybrene (Hanbio, Shanghai, China). Then, cells were cultured in media containing 2 μg/mL puromycin (Hanbio, Shanghai, China) for 24 h. qRT-PCR and Western blot analysis were utilized to analyze the transfection efficacy of NFIC at the mRNA and protein levels, respectively. The NFIC transfected cells were used for subsequent assays.

To overexpress G3BP1, colon cancer cells were infected with the lentivirus vector (Vector-G3BP1; Shanghai GenePharma Co., Ltd.) using 5 µg/ml polybrene (Hanbio Biotechnology Co., Ltd.) with an MOI of 10, and the infected cells were incubated with G401 (100 µg/ml) for 14 days at 37°C to establish the stably infected cells.
To silence β-catenin expression, colon cancer cells were infected with the lentivirus vector [short hairpin RNA (sh)-β-catenin; OriGene Technologies, Inc.] and puromycin (100 µg/ml) was applied to select the stably infected cells at 37°C for 14 days.
To knockdown G3BP1 expression, colon cancer cells were transfected with the small interfering (si)RNAs targeting G3BP1 (si-G3BP1; OriGene Technologies, Inc.) using Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The sequences were as follows: i) si-G3BP1-1, 5′-CCACACCAAGATTCGCCAT-3′; ii) si-G3BP1-2, 5′-GGAGATTCATGCAAACGTT-3′; iii) si-G3BP1-3: 5′-GGAGGAGTCTGAAGAAGAA-3′; and iv) si-NC: 5′-CCAAACCTTAGCGCACCAT-3′. Vector-NC, sh-NC and si-NC (OriGene Technologies, Inc.) were used as the negative controls for Vector-G3BP1, sh-β-catenin and si-G3BP1, respectively. After 48 h of transfection/infection, the cells were collected for subsequent experiments.

circPSD3 was packaged using lentivirus-GFP-puromycin, which was designed and synthesized by Hanbio. In brief, well-conditioned cells were seeded in 6-well plates at a density of 1 × 10⁵/mL. Lentiviral construct was transfected after cell confluence reached 50%–70%. After the cells were attached to the plates on the second day, the culture medium was discarded, and 1 mL fresh culture medium containing 8 μg/mL polybrene (Hanbio) was added for 30 min at 37°C in 5% CO₂. Then, 40 μL (3 × 10⁸ TU/mL) lentivirus was added to the culture medium for 48 h. The GFP expression efficiency was observed under an inverted fluorescent microscope. For the virus carrying the puromycin resistance gene, fresh culture medium containing 2 μg/mL puromycin was used to screen the stable strains.

The human GC cell line, SGC7901, was obtained from the Chinese Academy of Sciences Shanghai Cell Bank (Shanghai, China) and cultured in RPM-1640 (HyClone, Shanghai, China) supplemented with 10% fetal bovine serum (HyClone) in an incubator with 5% CO₂ at 37°C. The overexpression vector pLV-HOXA5-GFP-puro, the control vector pLV-GFP-puro and polybrene were obtained from Hanbio Technology (Shanghai, China). The SGC7901 cells were seeded at 1×10⁵/well in 6-well plates (1 ml/well). After 10 h, lentiviruses were added into the plates at a MOI of 40. polybrene was added at a final concentration of 5 µg/ml to each well. The culture media were replaced after 15 h. After 72 h, the cells were examined to determine the transduction efficiency under a fluorescence microscope (Olympus, Tokyo, Japan) and puromycin (Beyotime, Shanghai, China) was then added at a final concentration of 2 µg/ml to each well. After selection for 2 weeks, puromycin was removed and the transduced cells were used in further experiments.