Figure 3.

CC-885 Overcomes Volasertib Resistance in NSCLC Cells by Inducing Rapid Degradation of PLK1

(A) A549 cells with stable expression of Flag-PLK1 mutants (F183L and L59W) or PLK1 WT were cultured in a 96-well plate and treated with either DMSO, 50 nM volasertib, or 500 nM CC-885 for 24 h, and CCK-8 solution was added to each well and incubated for additional 4 h. The absorbance at 450 nm was then measured. Error bars indicate the means ± SD. ∗∗∗p < 0.001. (B) A549 cells with stable expression of Flag-PLK1 mutants (F183L and L59W) or PLK1 WT were treated with 1 μM CC-885 for 12 h, and cells were then harvested and subjected to western blot with the indicated antibodies. (C) Immunoblot analysis of A549 cells treated with the indicated doses of CC-885 for 12 h. Results are representative of three immunoblot analyses. (D) Time course of PLK1 protein levels in A549 cells treated with DMSO or 2 μM CC-885. (E) Time course of PLK1 mRNA levels in A549 cells treated with DMSO or 2 μM CC-885. Real-time PCR data are presented as the means ± SD (n = 3). (F) Immunoblot analysis of A549 cells treated with 100 μg/mL cycloheximide (CHX) and 100 nM CC-885 for the indicated periods. Cells were pretreated with 100 nM CC-885 for 2 h. Results are representative of three immunoblot analyses. Statistic results of western blotting analysis were obtained by ImageJ software and normalized to actin intensities. Error bars indicate the means ± SD. n = 3. (G) A549 cells were pretreated with 1 μM MLN4924 or 10 μM MG132 before the addition of 1μM CC-885 for 12 h. (H) A549 cells with stable expression of Flag-PLK1 mutants (F183L and L59W) or PLK1 WT were pretreated with 10 μM MG132 before the addition of 1 μM CC-885 for 12 h, and cells were then harvested and subjected to immunoprecipitation with Flag M2 beads followed by immunoblot analyses with the indicated antibodies. Results are representative of three immunoblot analyses. (I) Schematic diagram of the different FLAG-tagged PLK1 deletion mutants. (J) 293T cells transiently expressing either full-length FLAG-PLK1 or various FLAG- PLK1 mutants were treated with 1 μM CC-885 for 12 h, and cells were then harvested and subjected to western blot with the indicated antibodies. (K) A549 cells with stable expression of either full-length FLAG-PLK1 or FLAG- PLK1 1–584 were treated with 100 μg/mL CHX and 100 nM CC-885 for the indicated periods. Cells were then harvested and subjected to western blot with the indicated antibodies. (L) A549 cells with stable expression of Flag-PLK1 WT or PLK1 1–584 were pretreated with 10 μM MG132 before the addition of 1 μM CC-885 for 12 h, and cells were then harvested and subjected to immunoprecipitation with Flag M2 beads followed by immunoblot analyses with the indicated antibodies. (M) A549 cells with stable expression of Flag-PLK1 WT or PLK1 1-584 were cultured in a 96-well plate and treated with either DMSO, 50 nM volasertib, or 500 nM CC-885 for 24 h, and CCK-8 solution was added to each well and incubated for additional 4 h. The absorbance at 450 nm was then measured. Error bars indicate the means ± SD. ∗∗p < 0.01.