BCL-XL regulates the timing of mitotic apoptosis independently of BCL2 and MCL1 compensation

Mitotic catastrophe induced by prolonged mitotic arrest is a major anticancer strategy. Although antiapoptotic BCL2-like proteins, including BCL-XL, are known to regulate apoptosis during mitotic arrest, adaptive changes in their expression can complicate loss-of-function studies. Our studies revealed compensatory alterations in the expression of BCL2 and MCL1 when BCL-XL is either downregulated or overexpressed. To circumvent their reciprocal regulation, we utilized a degron-mediated system to acutely silence BCL-XL just before mitosis. Our results show that in epithelial cell lines including HeLa and RPE1, BCL-XL and BCL2 acted collaboratively to suppress apoptosis during both unperturbed cell cycle and mitotic arrest. By tagging BCL-XL and BCL2 with a common epitope, we estimated that BCL-XL was less abundant than BCL2 in the cell. Nonetheless, BCL-XL played a more prominent antiapoptotic function than BCL2 during interphase and mitotic arrest. Loss of BCL-XL led to mitotic cell death primarily through a BAX-dependent process. Furthermore, silencing of BCL-XL led to the stabilization of MCL1, which played a significant role in buffering apoptosis during mitotic arrest. Nevertheless, even in a MCL1-deficient background, depletion of BCL-XL accelerated mitotic apoptosis. These findings underscore the pivotal involvement of BCL-XL in controlling timely apoptosis during mitotic arrest, despite adaptive changes in the expression of other BCL2-like proteins.


SUPPLEMENTAL FIGURE LEGENDS
(A) mRNA expression of BCL2 versus BCL-XL.The levels of mRNA expression in 1,406 cell lines were retrieved from DepMap (depmap.org).The dataset 22Q2 Public was used.Pearson correlation: -0.287; p-value: 4.36x10 -28 . (B) Protein expression of BCL2 and BCL-XL in different cell lines.Expression of BCL2 and BCL-XL was detected with immunoblotting lysates from various asynchronously growing normal and cancer cell lines (upper panel).Band intensity was quantified and normalized to actin.Expression of BCL2 and BCL-XL in HeLa was arbitrarily set as 1. Cell origins: HeLa (cervical carcinoma), OVCA420 (ovarian carcinoma), MCF7 (mammary gland adenocarcinoma), MDA-MD-231 (breast adenocarcinoma), HCT116 (colorectal carcinoma), HT29 (colorectal adenocarcinoma), MIHA (immortalized hepatocytes), Hep3B (hepatocellular carcinoma), A549 (lung carcinoma), H1299 (lung carcinoma), U2OS (osteosarcoma), 293 (transformed human embryonic kidney), and RPE1 (hTERTimmortalized retinal pigment epithelial). (A) Generation of mAID BCL-XL KO cells with different number of AU elements.HeLa cells expressing mAID BCL-XL, tTA, and TIR1 were established using mAID BCL-XL constructs containing the indicated number of AU elements.Endogenous BCL-XL was concurrently disrupted with CRISPR-Cas9.After selection, a mixed population of cells was cultured in the presence or absence of DI.After 24 h, the cells were harvested and analyzed with immunoblotting.
(B) Generation of mAID BCL2 KO cells with different number of AU elements.HeLa cells stably expressing mAID BCL2, tTA, and TIR1 were established using mAID BCL2 containing the indicated number of AU elements.The endogenous BCL2 was at the same time disrupted with CRISPR-Cas9.After selection, a mixed population of the cells were cultured in either the presence or absence of DI.After 24 h, the cells were harvested and analyzed with immunoblotting.

Figure S3
. BCL-XL is expressed at a lower level than BCL2 and MCL1.
Lysates from untreated or IPTG-treated bacteria (two-fold dilution series) and BSA (two-fold dilution from 0.625 µg) were loaded onto SDS-PAGE and transferred to a membrane.Proteins on the membrane were visualized with Ponceau S staining, followed by blotting with antibodies against mAID.  A) Marginal acceleration of mitotic apoptosis in BCL2-deficient cells.Parental HeLa and mAID BCL2 KO cells were synchronized using a double thymidine procedure.Cells were trapped in mitosis using NOC and isolated by shake-off (t=0) before further incubated in NOC-containing medium.The cells were left untreated or treated with DI at the time of second thymidine release.Individual cells were tracked using live-cell imaging for 24 h (starting at 2 h after thymidine release) (n=50).Key: interphase (grey); mitosis (red); apoptosis (truncated bars).

Figure S1 .
Figure S1.Inverse relation between BCL2 and BCL-XL in cell lines.

Figure S2 .
Figure S2.Different expression of mAID-tagged proteins can be achieved by varying the number of AU elements.

(
C) Relative expression of BCL-W, BCL2, BCL-XL, and MCL1 in HeLa cells.The concentrations of endogenous BCL-W, BCL2, BCL-XL, and MCL1 in HeLa lysates were estimated by comparing their expression to mAID BCL-W, mAID BCL2, mAID BCL-XL, and mAID MCL1 using a mAID-H6 standard curve (in ng of the corresponding protein per µg of HeLa cell extracts.Mean of two independent experiments.

Figure
Figure S4.BCL-XL is a critical regulator of mitotic apoptosis.
(B) Acceleration of mitotic apoptosis in BCL-XL-deficient cells.HeLa and mAID BCL-XL KO cells were synchronized and arrested in mitosis as described in panel A. The cells were left untreated or treated with DI at the time of second thymidine release.Individual cells were tracked using live-cell imaging for 24 h, starting at 2 h after thymidine release (n=50).Key: interphase (grey); mitosis (red); apoptosis (truncated bars).

Figure
Figure S5.BCL-XL inhibits mitotic apoptosis in RPE1 cells.(A)Conditional depletion of BCL-XL.RPE1 cells expressing mAID BCL-XL, tTA, and TIR1 were generated.Endogenous BCL-XL was at the same time disrupted using CRISPR-Cas9.The cells were cultured in the presence or absence of DI and harvested at the indicated time points.Lysates were prepared and analyzed with immunoblotting.Lysates from parental RPE1 were loaded to show the expression of endogenous BCL-XL.Equal loading of lysates was confirmed by immunoblotting for actin.(B)BCL-XL is an inhibitor of mitotic apoptosis in RPE1.mAID BCL-XL KO RPE1 cells were incubated with DI and/or NOC as indicated.After 10 h, the cells were harvested and analyzed with immunoblotting.

(
C) Acceleration of mitotic apoptosis in BCL-XL-deficient RPE1.mAID BCL-XL KO RPE1 cells were incubated with NOC in the presence or absence of DI.Individual cells were tracked using live-cell imaging.Dot plots represent the elapsed time between mitotic entry and apoptosis (n=48; mean-/+SEM).The duration of mitotic arrest is plotted using Kaplan-Meier estimator.Mann-Whitney test: ***p<0.001.Raw data for individual cells are shown in the lower panel.

Figure S6 .
Figure S6.Predominance of BAX in mitotic apoptosis triggered by silencing BCL-XL.Parental mAID BCL-XL KO and mAID BCL-XL KO cells lacking BCL2, BAX, or BAK were synchronized and arrested in mitosis as described in Fig 4A.The cells were either left untreated or incubated with DI at the time of the second thymidine release.Individual cells were tracked using live-cell imaging for 24 h, starting at 2 h after thymidine release (n=50).Key: interphase (grey); mitosis (red); apoptosis (truncated bars).