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. 2020 Jun 23;18:215–225. doi: 10.1016/j.omto.2020.06.013

Figure 2.

Figure 2

CRBN, but Not GSPT1, Mediated the Synergistic Effect of Volasertib and CC-885

(A) Cell cycle distribution of A549 CRBN−/− cells treated with 50 nM volasertib, 50 nM CC-885, or both for 24 h. (B) Flow cytometry demonstrated the effects of 50 nM volasertib and the combination of 10 or 50 nM CC-885 with 50 nM volasertib on A549 CRBN−/− cells for 48 h. Error bars indicate the means ± SD. (C) A549 CRBN−/− cells were cultured in a 96-well plate treated with indicated doses of volasertib for 24 h, and CCK-8 solution was added to each well and incubated for an additional 4 h. The absorbance at 450 nm was then measured. Error bars indicate the means ± SD. (D) Morphological changes of the indicated doses of volasertib on A549 CRBN−/− cells for 24 h. Cells were imaged using a phase-contrast microscope. (E) A549 cells were infected with lentiviral vectors expressing control shRNA or any of the three GSPT1-specific shRNAs. Four days after infection, cells were collected for immunoblot analysis with the indicated antibodies. (F) A549 cells infected with lentiviral vectors expressing control shRNA or any of the three GSPT1-specific shRNAs for 4 days were cultured in a 96-well plate and treated with either DMSO or 50 nM volasertib for 24 h, and CCK-8 solution was added to each well and incubated for an additional 4 h. The absorbance at 450 nm was then measured. Error bars indicate the means ± SD. (G) A549 cells were treated with 50 nM volasertib, 50 nM CC-885, or both for 24 h. Cells were harvested and subjected to western blot with the indicated antibodies. (H) A549 cells were transfected with Flag-GSPT1 WT or MUT (G575N) for 36 h, and 1μM CC-885 was added for an additional 8 h. Cells were then harvested and subjected to western blot with indicated antibodies. (I) A549 cells transfected with Flag-GSPT1 WT or Flag-GSPT1 MUT (G575N) for 36 h were cultured in a 96-well plate and treated with either DMSO or 50 nM volasertib for 24 h, and CCK-8 solution was added to each well and incubated for an additional 4 h. The absorbance at 450 nm was then measured. Error bars indicate the means ± SD. ∗∗∗p < 0.001.