Twist1 sirna

Human GBM-derived ECs were transfected with siRNAs targeting Twist1 (Thermo Fisher Scientific, s14523), Snail (Thermo Fisher Scientific, s13185), Satb1 (Thermo Fisher Scientific, 106912), or control siRNA (Thermo Fisher Scientific, 4390843) using Lipofectamine 2000 Transfection Reagent (Life Technologies, 11668-019) in serum-free Opti-MEM medium (Gibco, 31985-070) for 12 hours, followed by incubation with serum-supplemented medium for 48 hours. For in vivo treatment, cells were incubated with LNPs containing Twist1 siRNA (Thermo Fisher Scientific, 69856) or control siRNA (Thermo Fisher Scientific, 4404020).

Twist1 siRNA and scrambled negative control were purchased from Bioneer (Daejeon, Korea). Twist1 siRNA #1 sense, 5ʹ-CUG AAC AGU UGU UUG UGU U-3ʹ; antisense, 5ʹ-AAC ACA AAC AAC UGU UCA G-3ʹ; #2 sense, 5ʹ-GGA CCC AUG GUA AAA UGC A; antisense, 5ʹ-UGC AUU UUA CCA UGG GUC C; #3 sense, 5ʹ-GCG CUU UCU UUU UGG ACC U; antisense, 5ʹ-AGG UCC AAA AAG AAA GCG C; scrambled negative control sense, 5ʹ-CCU ACG CCA CCA AUU UCG U-3ʹ; antisense, 5ʹ-ACG AAA UUG GUG GCG UAG G-3ʹ. A mixture of 100 nM siRNA and 5 μl Lipofectamine 2000 (Thermo Fisher Scientific Inc, MA, USA) was incubated in serum-free media for 30 min at RT. After incubation, the mixture was added to SK-Hep1 cells for siRNA transfection.