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. 2021 Mar 26;7(13):eabe1682. doi: 10.1126/sciadv.abe1682

Fig. 3. CPAB CMPX-321A induces BAK-mediated apoptosis.

Fig. 3

(A) Flow cytometric histograms showing overlay plots of NCI-H929 cells treated with control CPAB CMPX-322M (green) and anti–MCL-1 CPAB CMPX-321A (red) that have been stained with an antibody specifically recognizing activated BAK (n = 2). (B) Percentage of BAK-activated–positive NCI-H929 cells following CPAB treatment with anti–MCL-1 CPAB CMPX-321A (red) and control CPAB CMPX-322M (green). CMPX-321A induces a dose-dependent BAK activation. The control CPAB is not inducing BAK activation and is comparable to nontreated cells (n = 2; error bars represent the SEM). (C) Two-dimensional dot plots showing SUD-HL-4 cells after 6 hours of treatment with vehicle, CMPX-322M (2.5 to 5 μM), or CMPX-321A (2.5 to 5 μM) after annexin V fluorescein isothiocyanate (FITC)/PI staining. These plots show four regions corresponding to early apoptotic cells (PI-/FITC+; Q1), late apoptotic cells (PI+/FITC+; Q2), necrotic cells (PI+/FITC-; Q3), and viable cells (PI-/FITC-; Q4). A higher percentage of apoptotic cells are observed after CMPX-321A treatment compared to the vehicle or CMPX-322M control. (D) Upon CMPX-321A treatment (5 μM), a statistically higher percentage of apoptotic cells is observed compared to the cells that were treated with 2.5 and 5 μM of the control CMPX-322M [P = 0.0167 and P = 0.02, respectively; one-way analysis of variance (ANOVA)] *: P ≤ 0.05. **, P ≤ 0.01. (E) Bar charts showing the mean percentage of necrotic, dead, apoptotic, and healthy SU-DHL-4 cells after 6 hours of vehicle, CMPX-322M, and CMPX-321A treatment (n = 3; error bars represent the SEM).