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. 2021 Sep 3;11(19):9180–9197. doi: 10.7150/thno.60503

Figure 6.

Figure 6

Co-targeting of BCL-XL and CDK1/2/4 induces DNA damage, interferes with survival signals, and triggers apoptosis. (A) MMTV-R26Met cells were treated for 12 h with either A1155463 (A11: 1 µM; BCL-XL inhibitor), R547 (3 µM; CDK1/2/4 inhibitor) or Adavosertib (Adav: 3 µM; WEE1 inhibitor), alone or in combination, then subjected to western blot analysis. Note that R547 alone increased DNA damage in the MGT9 cell line, which exhibits the highest p53 levels as we reported in 4. We underline that the ACTIN panels are the same as in Figure 5 as western blots were performed simultaneously. (B) Densitometric analysis (using Image J) of western blots depicting protein levels. Mean values obtained with the three MGT cell lines are shown as fold of control (untreated cells). (C) Graphs depicting changes in levels of anti-apoptotic proteins (XIAP and MCL-1) as well as apoptosis markers (cleaved Caspase 3 and cleaved Caspase 7) in the four tumorigenic MMTV-R26Met cell lines following treatment with the indicated drugs, based on the RPPA analysis (Table S9). (D-F) DNA damage and mitotic catastrophe analysis. MGT11 cells were treated or not with A1155463 (0.3 µM), R547 (3 µM), Adavosertib (3 µM), or in combination. (D) Immunostaining with anti-γH2AX antibodies (to assess DNA damage) was performed after 12 h of treatment. The violin plot depicts the number of cells according to their γH2AX staining intensity. Representative images of γH2AX immunostaining (red) are shown on the right. (E) Cells treated for 16 h with the indicated drugs were immunostained with anti-pH3 antibodies. The graph reports the percentage of cells in mitosis (pH3-positive cells) versus the total number of cells. Representative images of pH3 immunostaining (red) are shown on the right. (F) Histogram reporting the number of mitotic catastrophe, (revealed by anti-pH3/α-Tubulin (microtubules) immunostaining) in treated cells. Mitotic catastrophe was analysed in cells in metaphase and anaphase among the pH3-positive cells. In contrast to WEE1 targeting (with Adavosertib), as previously reported 4, R547 does not induce mitotic catastrophe. In all experiments, DAPI was used to counterstain the nuclear DNA. Three independent experiments were performed. For multiple comparisons, statistical significance was assessed by One-way ANOVA followed by Tukey test. ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. Scale bar: 50 µm.