RNAi was performed as previously described [43 (link)]. Briefly, oligonucleotides of siRNA for thymidylate synthase, ANT2, and PHB2 were obtained from Dharmacon (Lafayette, CO, USA). The following siRNAs were used: siTS #1 (J-004717-07; ON-TARGET plus Human TYMS (7298) siRNA), 5′-UGGGAGAUGCACAUAUUUA-3′; siTS #2 (J-004717-08; ON-TARGET plus Human TYMS (7298) siRNA), 5′-UCACAUCGAGCCACUGAAA-3′; siANT2 #1 (D-007486-03; siGENOME Human SLC25A5 (292) siRNA), 5′-CUGCAGAUAAGCAAUACAA-3′; siANT2 #2 (D-007486-04; siGENOME Human SLC25A5 (292) siRNA), 5′-GCAGAUAAGCAAUACAAAG-3′; siPHB2 #1 (J-018703-07; ON-TARGET plus Human PHB2 (11331) siRNA), 5′-CAUCAAACUUCGCAAGAUU-3′; siPHB2 #2 (J-018703-08; ON-TARGET plus Human PHB2 (11331) siRNA), 5′-CAGAAUAUCUCCAAGACGA-3′. The following negative control siRNAs were used: ON-TARGET plus Non-Targeting siRNA #3 (D-001810-03), 5′-UGGUUUACAUGUUUUCUGA-3′; siGENOME Non-Targeting siRNA #5 (D-001210-05), 5′-UGGUUUACAUGUCGACUAA-3′. Cells were transfected with 10 nM siTS and siPHB2 or 30 nM siANT2 using Lipofectamine RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Watanabe M., Yamada Y., Kurumida Y., Kameda T., Sukeno M., Iizuka-Ohashi M., Sowa Y., Iizumi Y., Takakura H., Miyamoto S., Sakai T, & Mutoh M. (2021). Rabdosianone I, a Bitter Diterpene from an Oriental Herb, Suppresses Thymidylate Synthase Expression by Directly Binding to ANT2 and PHB2. Cancers, 13(5), 982.
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