Fig 2: Gene expression in CRC-82 and immunophenotypic levels of antiapoptotic molecules in the organoids generated. (A) BCL-2, BCL-XL, and BCL-W in primary human CRCs (#82-Tumor) and its corresponding normal tissue (#82-Normal). (B i–iii) Immunohistochemical staining of BCL-2, BCL-XL, and BCL-W in organoids (ORC-82). All images are taken at 20× magnification (scale bar: 50 µm). The statistical significant values p ≤ 0.05 are denoted by asterisk *.

Fig 3: EA.hy926 and HUVECs demonstrate differential protein expression of the BCL-2 family following DOX treatment. Both EA.hy926 cells and HUVECs were treated with 0.5 µM DOX for 24 h. Three days post DOX exposure, cells were harvested and total protein was extracted. Thereafter, protein expression levels of the BCL-2 family including anti-apoptotic members (BCL-2, BCL-xL, and BCL-W) and pro-apoptotic members (BAK and BAX) in (A) EA.hy926 cells and (B) HUVECs were measured via Western blotting (n = 4–6). Representative images of Western blots are shown. Values were normalized to α-tubulin and expressed relative to control cells. Values are presented as means ± SEM. Data were analyzed by unpaired two-tailed t-test. Note: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig 4: Immunofluorescence staining of antiapoptotic molecules in organoid OCR-82 after ABT-263 treatment. Panel (A) shows expression of BCL-2 (green); panel (B) shows expression of BCL-W (red); and panel (C) shows expression of BCL-XL (green) in DMSO control treatment (ORC-82-DMSO) and after ABT-263 treatment (ORC-82-ABT-263). Nuclei (blue) were stained by DAPI; quantification of immunofluorescence signals are represented in bar diagrams; and all images are at 10× magnification (scale bar: 100 µm).

Fig 5: Bcl-xL is a major suppressor of alisertib–danusertib-induced apoptosis in HCT-116 cells.A, single clones of Bcl-2 KO, Bcl-w KO, Bcl-xL KO, and Mcl-1 KO cells were generated from HCT116 cells by CRISPR–Cas9. Cells were harvested, and the whole-cell lysate was used to examine KO by Western blot. B, HCT116 WT and single KO cells established in (A) were treated with alisertib (1 μM) and danusertib (1 μM) for 24 h, and cells were harvested and the whole-cell lysate was used for Western blot analysis. C, same cell lines and treatments as described in (B) were subjected to Hoechst staining for quantification of apoptotic cells. Mean and SD values were shown on the graph, n = 3. Representatives of at least three independent experiments were shown for Western blots. Two-way ANOVA was used to test the significance, p < 0.0001 was defined as significant and indicated with ∗∗∗∗.