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CRC cell lines were seeded into 6-well plates at a density of 1 × 105 cells per well and cultured under standard conditions in a cell culture incubator at 37 °C until they reached 60% confluence. Following the guidelines outlined in the Lipofectamine 2000 manual, serum-free medium containing the appropriate plasmids and vectors was introduced into the 6-well plates. Transfected cells were harvested at 24, 48, or 72 h post-transfection, and the effectiveness of plasmid expression was evaluated through qRT-PCR for subsequent analyses.
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