Extended Data Fig. 7: GS depletion does not affect the interaction of NUP88-RAE1-NUP98, but augments the interaction of NUP88 and CDC20.
From: Glutamine synthetase licenses APC/C-mediated mitotic progression to drive cell growth
a, Co-IP experiments conducted with the lysate of H460 cells as indicated using anti-RAE1, anti-NUP98, and anti-NUP88 antibodies. b, Expression of PLK1 in H460 cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C. c, H460 cells transduced with control shRNA or GS-targeted shRNA were arrested at mitotic prometaphase (PrM), metaphase (Met), or anaphase (Ana) by indicated treatment. Cell lysate was collected for Co-IP experiments using an anti-NUP88 antibody. d, Content of the purified proteins used in Fig. 5b. e, Co-IP experiments conducted with the lysate of H460 cells of b using an anti-NUP88 antibody. Cells were treated with nocodazole for 18 hours before M-phase cells harvested for co-IP experiments. f, Upper: expression of GS in H460 cells transduced with control shRNA, shGS-1 in the presence of 0.5 mM or 2 mM glutamine. Lower: H460 cells transduced with control shRNA or GS-targeted shRNA were cultured with 0.5 mM or 2 mM glutamine for 8 hours then treated with nocodazole for 18 hours. Floating cells were collected and treated with MG132 for another hour. Cell lysate was collected for co-IP experiments using an anti-CDC27 antibody. g, In vitro pull-down experiments conducted with the mixture of the purified Myc-CDC20, HA-CDC27 and different dosages of V5-NUP88 protein using an anti-HA antibody. h, Co-IP experiments conducted with the lysate of H460 cells cultured in 0.5 mM or 2 mM glutamine using an anti-NUP88 antibody. i. Percentage of G2/M cells of H460 cells transduced with control shRNA or GS-targeted shRNA after released from nocodazole treatment as indicated time points. (n=3 independent biological replicates). j, Expression of CDC20 in H460 cells transduced with control shRNA, control shRNA combined with overexpression of CDC20, shGS-1, and shGS-1 combined with overexpression of wild-type CDC20. k, The fold change of polyploid cells in H460 cells of j. (n=3 independent biological replicates). Data were presented as mean±s.e.m. Unpaired, two-tailed t-tests were applied for i and k. *: p<0.05, **: p<0.01.
