The appropriate recombinant SW48 cells were plated in a 10-cm plate in DMEM supplemented with 10% FBS 24 hours before transfection. The following day, cells were transfected with siRNAs against NF1 (2 μg) or control siRNA (2 μg) using Lipofectamine 2000. For EGFR knockdown, cells were plated in a 96-well format in 100 μl of Opti-MEM (10% FBS) with 0.1 μg of siRNA mixed with 0.5 μl of Lipofectamine 2000 per well. Twenty-four hours after EGFR siRNA delivery, cells were treated with cetuximab for 48 hours, and proliferation was measured by MTT assay. Silencer Select siRNAs were purchased from Thermo Fisher Scientific. Silencer Select-NF1 (s56534) was composed of pooled RNAs targeting exons 2, 10, 16, 18, and 19 in the NF1 mRNA. Silencer Select-EGFR (s565) was composed of pooled siRNA targeting five unique sequences within exon 2 of the EGFR mRNA. Silencer Select Control siRNA (4390843) was used as negative control. All siRNAs were reconstituted in ribonuclease-free molecular-grade water upon arrival from vendor at a concentration of 5 mM.

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