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. 2020 Feb 14;9:e53447. doi: 10.7554/eLife.53447

Figure 4. Proteasome inhibition suppresses ETO-induced genome instability and improves cell survival.

Figure 4.

(A) Example of mitotic spreads from WT primary splenic B-cells treated for 2 hr in G1 with 50 µM ETO ± 1 hr 5 µM BTZ pre-treatment, followed by 24 hr recovery in drug free medium prior to harvesting metaphases. Colored arrows indicate the type of aberrations as quantified and described in B. (B) Analysis of chromosomal aberrations (Chromosome Breaks (yellow), Chromatid Breaks (orange), Fusions (blue), Radials (purple) and Fragments (green)). 50 metaphases were counted for each condition. (C) Example of mitotic spreads from WT primary splenic B-cells treated for 2 hr in G1 with 50 µM ETO ± 1 hr 10 µM MG132 pre-treatment, and then harvested 24 hr after washout for Spectral Karyotyping Analysis (SKY) (Left and right top panels). SKY reveals more complex fusions involving multiple chromosomes, compared to the fusions observable by DAPI staining only (Left and right bottom panels). (D) Analysis of chromosomal fusion determined by SKY analysis. Chromosome fusions were counted in 35 mitotic spreads per condition and were broken down by how many chromosomes were involved (1, two or greater than 3 (+)) per fusion event per cell. (E) Quantification of percent cells in S phase determined by FACS. WT primary splenic B-cells were treated in G1 for 2 hr with 50 µM ETO ± 1 hr 10 µM MG132 pre-treatment. 16 or 24 hr after washout, cells were pulsed for 30 min with EdU pulse an analyzed by FACS (16 hr NT vs MG132 p=0.012; 16 hr NT v ETO p=0.0485; 24 hr ETO vs pre-MG132 + ETO p=0.049; 24 hr ETO vs. post-MG132 p=0.46; statistical significance calculated by student T-test). (F) Viability of WT primary splenic B-cells as determined using the CellTiter-Glo luminescence assay 48 hr following 2 hr of 50 µM ETO treatment ±pre treatment, co-treatment, or post-treatment with 5 µM BTZ (BTZ v ETO p=0.0006, ETO v pre-BTZ + ETO p=0.015, statistical significance calculated by student T-test). During treatment cells were in the G1 phase of the cell cycle.