Eight phosphorothioate-modified ASOs targeting TROJAN were designed and purchased from BioSune in Shanghai, China: ASO-1, 5′-mUmUmAmGmCAGCTTGAAGCmCmAmGmGmU-3′; ASO-2, mCmAmAmUmCTGCTTAAGAGmAmCmUmGmC; ASO-3, mUmGmAmAmCAGTTTCAAGAmGmAmUmGmA; ASO-4, mAmCmUmUmGTGGCAATAGTmGmAmAmCmA; ASO-5, mGmGmAmCmAGAGAGTGGGCmUmGmAmUmA; ASO-6, mAmGmGmAmCAGAGAGTGGGmCmUmGmAmU; ASO-7, mGmGmAmUmTATAACCTGGGmAmGmCmAmA; ASO-8, mCmUmGmCmATCCTTTGGTGmUmUmAmCmA; ASO-4 (18-mer), mCmUmUmGTGGCAATAGTmGmAmAmC; ASO-4 (16-mer), mUmUmGTGGCAATAGTmGmAmA; ASO-4 (14-mer), mUmGTGGCAATAGTmGmA; ASO-4 (13-mer), mUmGTGGCAATAGmUmG.

An ASO not matching any known human transcripts (Ionis 141923) was used as the negative control (ASO-Ctrl, mCmCmUmUmCCCTGAAGGTTmCmCmUmCmC). For in vitro ASO delivery, 1 × 105 MDA-MB-231 LM2 cells were seeded in six-well plates and transfected with 1.4 μg of ASO premixed with 5.6 μl of Lipofectamine 2000, and RNA was isolated after 24 hours. For the in vitro proliferation assay, 1 × 103 MDA-MB-231 LM2 cells were seeded in 96-well plates and transfected with 0.35 μg of ASO premixed with 0.35 μl of Lipofectamine 2000. For the in vitro free uptake assay, 1 × 105 MDA-MB-231 LM2 cells were seeded in six-well plates and transfected with ASOs at various concentrations (0, 0.5, 1, 2.5, 5, and 10 μM) without a transfection reagent, and RNA was isolated after 24 hours. For in vivo ASO delivery, ASOs were delivered by intraperitoneal injection at a dose of 25 mg/kg (lung metastasis xenograft model) or 50 mg/kg (mammary fat pad xenograft model). PBS was injected as the control.

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