mcl1 inhibitor s63458  (MedChemExpress)

Journal: Cell Death Discovery

Article Title: Mitochondrial priming and response to BH3 mimetics in “one-two punch” senogenic-senolytic strategies

doi: 10.1038/s41420-025-02379-y

Figure Lengend Snippet: A Top . Representative images ( n = 3) of SA-β-gal staining in A549 lung cancer cells treated with senescence-inducing concentrations of bleomycin, alisertib, doxorubicin or palbociclib for 7 days (scale bar: 500 μm for proliferative/untreated cells; 200 μm for TIS cells). Bottom left . Expression levels of phospho-RB Ser807/Ser811 and p21 WAF1/Cip1 were detected by immunoblotting in whole cell lysates of proliferative (untreated) and TIS A549 cancer cells (7-day) using specific antibodies and β-actin/GAPDH as loading controls. The figure shows a representative immunoblot from multiple ( n = 3) independent experiments (PLB Palbociclib, DOX Doxorubicin, ALI Alisertib, BLEO Bleomycin). Bottom right . Representative flow cytometry plots showing the gating of the cell cycle distribution of proliferative (untreated) and TIS phenotypes. The histogram shows the percentage (mean ± S.D., n = 3) of cells in the four cell cycle phases as a function of the treatment condition. B Top . Representative microphotographs of proliferative (untreated) and TIS cancer cells labeled with IncuCyte ® Annexin V Green reagent 3 days after harvest and reseeding. Bottom . TIS cancer cells and proliferative (untreated) cells were reseeded into 96-well plates (6000 cells/well and 2000 cells/well, respectively) and cultured with increasing concentrations of ABT-263/navitoclax. After 5 days, cell viability was measured using the alarmarBlue™ assay. Shown are representative images of alamarBlue™-based cell viability assays showing changes in the number of metabolically active cells in response to serial dilutions of BH3 mimetics and dose-response curves for ABT-263/navitoclax in proliferative (untreated) and TIS A549 lung cancer cells (untreated = 100%). Bar graphs of the IC 50 values determined as the μmol/L concentrations of ABT-263/navitoclax required to decrease cell viability by 50%, and the senolytic indexes obtained by dividing the IC 50 values of ABT-263/navitoclax in proliferative A549 cells by those obtained in TIS A549 cells. Data represent the mean ± S.D. of ≥3 independent experiments performed in triplicate. Statistically significant differences (ANOVA analysis) between proliferative and TIS phenotypes means are shown. n.s. not statistically significant. C TIS cancer cells and proliferative (untreated) cells were replated into 96-well plates (6000 cells/well and 2000 cells/well, respectively) and cultured with increasing concentrations of ABT-199/venetoclax, A1331852 or S63845. After 5 days, cell viability was measured using the alarmarBlue™ assay. Shown are representative images of alamarBlue™-based cell viability assays showing changes in the number of metabolically active cells in response to serial dilutions of BH3 mimetics and dose-response curves for ABT-199/venetoclax, A1331852 or S63845 in proliferative (untreated) and TIS A549 lung cancer cells (untreated = 100%). See also Table .

Article Snippet: To further confirm that the heterogeneous responses of TIS cells to BH3 senolytics were not limited to ABT-263/navitoclax, we evaluated the senolytic sensitivity of A549 TIS cells using a broader toolkit of BH3 mimetics, namely the BCL2-specific inhibitor ABT-199/venetoclax, the BCL-X L -specific inhibitor A-1331852, and the MCL1-specific inhibitor S63845.

Techniques: Staining, Expressing, Western Blot, Flow Cytometry, Labeling, Cell Culture, Metabolic Labelling

Journal: Cell Death Discovery

Article Title: Mitochondrial priming and response to BH3 mimetics in “one-two punch” senogenic-senolytic strategies

doi: 10.1038/s41420-025-02379-y

Figure Lengend Snippet: Sensitivity of TIS A549 lung cancer cells to BH3 mimetics.

Article Snippet: To further confirm that the heterogeneous responses of TIS cells to BH3 senolytics were not limited to ABT-263/navitoclax, we evaluated the senolytic sensitivity of A549 TIS cells using a broader toolkit of BH3 mimetics, namely the BCL2-specific inhibitor ABT-199/venetoclax, the BCL-X L -specific inhibitor A-1331852, and the MCL1-specific inhibitor S63845.

Techniques:

Journal: Cell Death Discovery

Article Title: Mitochondrial priming and response to BH3 mimetics in “one-two punch” senogenic-senolytic strategies

doi: 10.1038/s41420-025-02379-y

Figure Lengend Snippet: A549 lung cancer cells were treated with senescence-inducing concentrations of bleomycin, alisertib, doxorubicin, or palbociclib for 7 days. Senescent cells and proliferative (untreated) cells were replated in 12-well plates (100,000 cells/well and 50,000 cells/well, respectively) and cultured with increasing concentrations of ABT-263/navitoclax, A13318512, ABT-199/venetoclax, or S63845. After 5 days, cell growth and senescence were monitored by crystal violet staining ( left panels ) and SA-β-gal staining ( right panels ). Shown are representative images from three technical replicates. Scale bar = 200 μm. Bottom left . BH3 mimetic/anti-apoptotic protein interaction pattern and senolytic sensitivity profile of TIS phenotypes. ABT-263/navitoclax inhibits BCL-2 and BCL-X L , A13318512 inhibits BCL-X L only, ABT-199/venetoclax inhibits BCL-2 only, and S63845 inhibits MCL1 only. Bottom right . Summary of the responses of A549 TIS cancer cells to BH3 mimetics.

Article Snippet: To further confirm that the heterogeneous responses of TIS cells to BH3 senolytics were not limited to ABT-263/navitoclax, we evaluated the senolytic sensitivity of A549 TIS cells using a broader toolkit of BH3 mimetics, namely the BCL2-specific inhibitor ABT-199/venetoclax, the BCL-X L -specific inhibitor A-1331852, and the MCL1-specific inhibitor S63845.

Techniques: Cell Culture, Staining

Journal: Cell Death Discovery

Article Title: Mitochondrial priming and response to BH3 mimetics in “one-two punch” senogenic-senolytic strategies

doi: 10.1038/s41420-025-02379-y

Figure Lengend Snippet: A Top . Both the level of mitochondrial priming—the proximity to the mitochondrial apoptotic threshold that determines the ability of TIS mitochondria to initiate apoptosis in response to BH3 peptides—and the nature of apoptotic blockade in TIS cancer cells—the distinct patterns of dependence on pro-survival BCL-2 proteins in the BCL2/BH3 interactome—were assessed using the plate-based JC-1 BH3 profiling. After proliferative (untreated) and TIS cancer cells are permeabilized with digitonin to allow BH3 peptides to diffuse into the cells and interact with intact mitochondria, the loss of JC-1 red fluorescence caused by depolarization of the mitochondrial transmembrane potential (a surrogate for the endpoint MOMP) allows real-time kinetic measurements of the loss of mitochondrial integrity. Primed mitochondria respond more robustly to both activator and sensitizer BH3 peptides and are more susceptible to apoptosis. The pattern of response to BH3 peptides can also identify the functional dynamics between pro- and anti-apoptotic proteins in maintaining cell survival in the TIS phenotypes (e.g., defects in pro-apoptotic signaling or increased addiction to anti-apoptotic proteins). Cell viability analysis using a panel of BH3 mimetics (ABT-263, ABT-199, A13318512, S63845) was used to assess senolytic indexes (SI), which were defined as the ratio of IC 50 values of proliferative cancer cells to IC 50 values of TIS cancer cells. Bottom . Apoptotic blocks based on BH3 profiling. After exposure to individual activator or sensitizer BH3 peptides, BH3 profiling can distinguish three major blocks (stop signs) through which cells can avoid apoptosis, namely: primed, unprimed-competent, and unprimed-incompetent. Bottom . Patterns of interaction between the anti-apoptotic proteins present in cells ( columns ) and the pro-apoptotic synthetic peptides or drugs ( rows ) used in the BH3 profiling assay and the BH3 mimetics cell viability toolkit. BIM, BID, and PUMA inhibit all the inhibitors and are pan-sensitizers. PUMA (shown in red rows) can act as an activator of BAX and BAK. Orange colors highlight those peptides/drugs that inhibit the BCL-2 protein, including BMF and BAD, Blue rows highlight the dependency on mantle cell lymphoma (MCL)-1, whereas NOXA inhibits MCL-1 and BFL-1. Green indicates BCL-X L dependency, as HRK is primarily a BCL-X L inhibitor, but can also inhibit other anti-apoptotic proteins with lower affinity ( A and B , created with Biorender.com ) . BH3 profiling was performed to measure mitochondrial depolarization in proliferative (untreated) and TIS A549 lung cancer cells exposed to activator ( B ) and sensitizer ( C ) BH3 peptides. Figure shows heat maps of % mitochondrial depolarization caused by increasing concentrations of the activator (BIM, BID, PUMA) and sensitizer (BMF, BAD, NOXA, HRK) peptides in proliferative (untreated) and palbociclib, doxorubicin, alisertib, and bleomycin TIS cells. Data shown are the mean of ≥3 independent experiments using three technical replicates for each peptide. Graphs represent the means ( columns ) ± S.E.M. ( bars ) of ≥3 independent experiments BH3 peptide EC 50s (μmol/L) in proliferative and palbociclib, doxorubicin, alisertib, and bleomycin TIS cells. Statistically significant differences (ANOVA analysis) between proliferative and TIS phenotypes means are shown. n.s. not statistically significant. D Global heat maps of % mitochondrial depolarization caused by selected concentrations of activator and sensitizer peptides in A549 lung cancer cells. Samples are ordered according to depolarization by the HRK peptide. Data shown are the mean of ≥3 independent experiments with three technical replicates for each peptide.

Article Snippet: To further confirm that the heterogeneous responses of TIS cells to BH3 senolytics were not limited to ABT-263/navitoclax, we evaluated the senolytic sensitivity of A549 TIS cells using a broader toolkit of BH3 mimetics, namely the BCL2-specific inhibitor ABT-199/venetoclax, the BCL-X L -specific inhibitor A-1331852, and the MCL1-specific inhibitor S63845.

Techniques: Fluorescence, Functional Assay

Journal: Cell Death Discovery

Article Title: Mitochondrial priming and response to BH3 mimetics in “one-two punch” senogenic-senolytic strategies

doi: 10.1038/s41420-025-02379-y

Figure Lengend Snippet: A Left . BH3 profiling was performed to measure mitochondrial depolarization in proliferative (untreated) and TIS LoVo colon cancer cells exposed to activator and sensitizer BH3 peptides. Figure shows heat maps of % mitochondrial depolarization caused by increasing concentrations of the activator (BIM, BID, PUMA) and sensitizer (BMF, BAD, NOXA, HRK) peptides in proliferative (untreated) and palbociclib and bleomycin TIS cells. Data shown are the mean of ≥3 independent experiments using three technical replicates for each peptide. Graphs represent the means ( columns ) ± S.E.M. ( bars ) of ≥3 independent experiments BH3 peptide EC 50s (μmol/L) in proliferative (untreated) and palbociclib and alisertib TIS cells. Statistically significant differences (ANOVA analysis) between proliferative and TIS phenotypes means are shown. n.s. not statistically significant. Right . Figure shows global heat maps of % mitochondrial depolarization caused by selected concentrations of activator and sensitizer peptides in LoVo colon cancer cells. Samples are ordered according to depolarization by the BIM peptide. Data shown are the mean of ≥3 independent experiments with three technical replicates for each peptide. B TIS cancer cells and proliferative (untreated) cells were replated into 96-well plates (7,000 cells/well and 4,000 cells/well, respectively) and cultured with increasing concentrations of ABT-263/navitoclax, A1331852, ABT-199/venetoclax, or S63845. After 5 days, cell viability was measured using the alarmarBlue™ assay. Shown are representative images of alamarBlue™-based cell viability assays showing changes in the number of metabolically active cells in response to serial dilutions of BH3 mimetics and dose-response curves for ABT-263/navitoclax, A1331852, ABT-199/venetoclax, or S63845 in proliferative (untreated) and TIS LoVo cancer cells (untreated=100%). See also Table . C Top . LoVo colon cancer cells were treated with senescence-inducing concentrations of alisertib or palbociclib for 7 days. Senescent cells and parental (proliferative) cells were replated into 12-well plates (200,000 cells/well and 150,000 cells/well, respectively) and cultured with increasing concentrations of ABT-263/navitoclax, A1331852, ABT-199/venetoclax, or S63845. After 5 days, cell growth and senescence were monitored using crystal violet staining ( left panels ) and SA-β-gal staining ( right panels ). Shown are representative images from three technical replicates. Scale bar = 200 μm. Bottom . Summary of the responses of LoVo TIS cancer cells to BH3 mimetics.

Article Snippet: To further confirm that the heterogeneous responses of TIS cells to BH3 senolytics were not limited to ABT-263/navitoclax, we evaluated the senolytic sensitivity of A549 TIS cells using a broader toolkit of BH3 mimetics, namely the BCL2-specific inhibitor ABT-199/venetoclax, the BCL-X L -specific inhibitor A-1331852, and the MCL1-specific inhibitor S63845.

Techniques: Cell Culture, Metabolic Labelling, Staining

Journal: Cell Death Discovery

Article Title: Mitochondrial priming and response to BH3 mimetics in “one-two punch” senogenic-senolytic strategies

doi: 10.1038/s41420-025-02379-y

Figure Lengend Snippet: Sensitivity of TIS LoVo colon carcinoma cells to BH3 mimetics.

Article Snippet: To further confirm that the heterogeneous responses of TIS cells to BH3 senolytics were not limited to ABT-263/navitoclax, we evaluated the senolytic sensitivity of A549 TIS cells using a broader toolkit of BH3 mimetics, namely the BCL2-specific inhibitor ABT-199/venetoclax, the BCL-X L -specific inhibitor A-1331852, and the MCL1-specific inhibitor S63845.

Techniques:

Journal: Cell Death Discovery

Article Title: Long-term adaptation of lymphoma cell lines to hypoxia is mediated by diverse molecular mechanisms that are targetable with specific inhibitors

doi: 10.1038/s41420-025-02341-y

Figure Lengend Snippet: A Apoptosis was determined 24 h after exposure of the original lymphoma cell lines cultured under standard normoxic tissue culture conditions (blue) and HA cells cultured in uninterrupted hypoxia (red), 2-DG = 2-deoxy-glucose (inhibitor of glycolysis), IM-156 = inhibitor of oxidative phosphorylation, venetoclax, A1155463, and S63845 = inhibitor of BCL2, BCL-XL, and MCL1, respectively, TRAIL = Tumor necrosis factor alpha-related apoptosis-inducing ligand, copanlisib = pan-AKT inhibitor; cisplatin, cytarabine = genotoxic cytostatics; bortezomib = proteasome inhibitor; results represent means ± standard deviations (SD) of three independently implemented experiments; ( B ) western blot analysis of biological duplicates of HA cells (cultured in uninterrupted hypoxia) compared to the original lymphoma cell lines (cultured in standard normoxic tissue culture conditions); ( C ) immunoprecipitation (IP) of the HA lymphoma cells (cultured in uninterrupted hypoxia) compared to the original lymphoma cell lines (cultured in standard normoxic tissue culture conditions) with anti-BCL-XL antibody and subsequent detection of proapoptotic BIM and BAK bound on BCL-XL; increased amounts of proapoptotic BIM could be detected in total lysates of both HA HBL2 and HA Ramos cells (compared to the normoxic controls); increased amount of BIM was detected bound on BCL-XL; L= total lysates, IgG stands for lysates immunoprecipitated with polyclonal rabbit IgG; BCL-XL stands for lysates immunoprecipitated with anti-BCL-XL antibody; BIM-EL/L/S = extra-large / large / small BIM isoforms. Increased amount of proapoptotic effector BAK was detected bound on BCL-XL in HA Ramos cells. Proapoptotic BAX was not detected bound on BCL-XL in either tested cell line.

Article Snippet: Venetoclax, a BCL2 inhibitor, S63845, a MCL1 inhibitor, and A1155463, a BCL-XL inhibitor, and IM-156, an inhibitor of oxidative phosphorylation, were purchased from MedChemExpress (via Scintila s.r.o., Czech Republic).

Techniques: Cell Culture, Western Blot, Immunoprecipitation