Nup98 Silencing in MCF7 and PC3 Cells

MCF7 cells were a gift from S. Merajver’s laboratory (University of Michigan). PC3 cells were a stable cell line expressing cell cycle reporters hCdt1-mCherry and p27K-mVenus previously described (Takahashi et al., 2019 (link)). The cells were grown to 50-70% confluency in a 12-well plate or eight-well chamber slide. The cells were then transfected with 20 nM Nup98 siRNA or control siRNA using Lipofectamine RNAi MAX (Invitrogen), following the manufacturer's protocol. The cells were incubated with the indicated siRNA for 72 h, then harvested for fixation and staining or lysed for western blotting. siRNAs used were as follows: Silencer Select Negative Control No. 1 (4390843, ThermoFisher Scientific); Nup98-96 siRNA#1, Silencer Pre-designed siRNA (AM16708, ThermoFisher Scientific); Nup98-96 siRNA#2, Silencer Select Pre-designed siRNA (4392420, ThermoFisher Scientific); Nup98-96 siRNA#3, Nup98 siRNA (sc-43436, Santa Cruz Biotechnology). Cell lines were tested for mycoplasma routinely and were negative in June 2021. PC3 cells were authenticated prior to publication (Takahashi et al., 2019 (link)).