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. 2020 Sep 17;11(9):769. doi: 10.1038/s41419-020-02944-6

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Concentration-response curves of the DLBCL cell lines SU-DHL-4, KARPAS-422, SU-DHL-6, Ri-1, TOLEDO, OCI-LY-1, OCI-LY-18, and PFEIFFER treated with increasing concentrations of BDA-366. After 24 h the level of apoptosis was measured using flow cytometeric analysis in Annexin V-FITC/7-AAD stained cells. Data represented here are averages ± SEM of at least three independent experiment (N > 3). b Quantification of the expression levels of Bcl-2 in several cell lines (right) normalized to SU-DHL-4 (left). Correlation plot of the LD₅₀ values for BDA-366-induced cell death and different Bcl-2-family protein expression levels in distinct cell lines obtained via linear regression analysis (right). Data are presented as average ± SD of three independent experiments (N = 3). c Concentration-response curves of two DLBCL cell lines and two murine thymocytes respectively: HT, HT overexpressing Bcl-2 (HT Bcl-2), Wehi7.2 overexpressing (Wehi7.2 Bcl-2) and Wehi7.2. Cell lines were treated with increasing concentrations of BDA-366. After 24 h the level of apoptosis was measured using flow cytometric analysis in Annexin V-FITC/7-AAD stained cells. Data represented here are averages ± SEM of at least three independent experiment (N > 3). d Overexpression of Bcl-2 in primary human CLL cells by Bcl-2 mRNA transfection increases resistance to BDA-366 and venetoclax. Primary human CLL cells (n = 6) were transfected with control (Ctrl) or Bcl-2 mRNA, left in culture for 3 h to achieve maximal Bcl-2-protein expression, and were then exposed to BDA-366 (1 μM) or venetoclax (4 nM). The viability of the leukemic cells was determined after 20 h by Annexin V/PI staining. Statistical analysis was done with the paired t test for the comparison of the control and the venetoclax-treated cells, whereas because of non-normal distribution the Wilcoxon Signed Rank test was applied for the comparison of the BDA-366-treated cells.