Multi-Modality Apoptosis Assay Protocol

Alisertib (MLN8237) (catalog no.: S1133), danusertib (PHA-739358) (catalog no.: S1107), doxorubicin (adriamycin) HCl (catalog no.: S1208), and bortezomib (PS-341) (catalog no.: S1013) were purchased from SelleckChem and dissolved in dimethyl sulfoxide (DMSO). ABT-737 (catalog no.: 852808-04-9) and S63845 (catalog no.: 1799633-27-4) were purchased from ChemieTek and dissolved in DMSO. Camptothecin (catalog no.: 159732) and z-VAD-FMK (catalog no.: 03FK1090-CF) were purchased from MP Biomedicals and dissolved in DMSO. Human recombinant TRAIL was generated as previously described (51 (link)). 1,6-Bismaleimidohexane was purchased from Thermo Fisher Scientific (catalog no.: 22330). Annexin V-FITC was purchased from BioLegend (catalog no.: 640906). Propidium iodide (PI) solution was purchased from G-Biosciences (catalog no.: 786-1272).
Antibodies used for immunoblotting include anti-β-actin (Sigma–Aldrich; catalog no.: A5441), anti–caspase-3 (Santa Cruz Biotechnology; catalog no.: sc-56053), anti-GFP (Santa Cruz Biotechnology; catalog no.: sc-9996), anti-MCL-1 (Santa Cruz Biotechnology; catalog no.: sc-819), anti-BCL-2 (Cell Signaling Technology; catalog no.: 15071), anti-Bcl-xL (Cell Signaling Technology; catalog no.: 2762), anti-BCL-w (Cell Signaling Technology; catalog no.: 2724), anti-Bax (Cell Signaling Technology; catalog no.: 2772), anti-Bak (Cell Signaling Technology: catalog no.: 5023), anti-PUMA (Cell Signaling Technology; catalog no.: 12450), anti-Bid (36 (link)), anti-Bim-EL (Cell Signaling Technology; catalog no.: 2819), anti-BAD (Santa Cruz Biotechnology; catalog no.: sc-3044), anti-Noxa (Santa Cruz Biotechnology; catalog no.: sc-515840), anti-BIK (Cell Signaling Technology; catalog no.: 4592), anti–caspase-2 (Cell Signaling Technology; catalog no.: 2224), anti–caspase-8 (Cell Signaling Technology; catalog no.: 9746S), anti-p53 (Santa Cruz Biotechnology; catalog no.: sc-393), and anti-GFP (Santa Cruz Biotechnology; catalog no.: sc-459).
Secondary antibodies include anti-rabbit (Sigma–Aldrich; catalog no.: A6154) and antimouse (Jackson ImmunoResearch, Inc; catalog no.: 715-035-150).

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Li J., Chen C.H., O’Neill K.L., Fousek-Schuller V.J., Black A.R., Black J.D., Zhang J, & Luo X. (2023). Combined inhibition of aurora kinases and Bcl-xL induces apoptosis through select BH3-only proteins. The Journal of Biological Chemistry, 299(2), 102875.

Publication 2023

Abt 737 Actin Adriamycin Alisertib Annexin v fitc Antibodies Bcl 2 Bortezomib Camptothecin Caspase 2 Caspase 3 Caspase 8 Danusertib Dimethyl sulfoxide Doxorubicin Human Mln8237 Pha 739358 Propidium iodide Ps 341 Puma Rabbit S63845 Trail Z vad fmk

Corresponding Organization : University of Nebraska Medical Center

Top 5 similar protocols

Variable analysis

independent variables

Alisertib (MLN8237)
Danusertib (PHA-739358)
Doxorubicin (adriamycin) HCl
Bortezomib (PS-341)
ABT-737
S63845
Camptothecin
Z-VAD-FMK
Human recombinant TRAIL
1,6-Bismaleimidohexane

dependent variables

Cell viability, apoptosis, and other outcomes (not explicitly mentioned)

control variables

Dimethyl sulfoxide (DMSO) - used to dissolve the compounds

controls

Positive control: Human recombinant TRAIL
Negative control: Not explicitly mentioned

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