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. 2018 Jun 13;26(3):531–547. doi: 10.1038/s41418-018-0142-3

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© The Author(s) 2018

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. 6 — PLC inhibition also suppresses BIRD-2-triggered apoptosis in other DLBCL cell lines. a, b, c Quantitative analysis of at least 3 independent experiments detecting apoptosis in Annexin V-FITC/7-AAD-stained SU-DHL-6 (a), Karpas 422 (b), and RI-1 (c) cells treated with U73122, U73343, BIRD-2 (blue), venetoclax (green) or a combination of U73122/U73343 with BIRD-2/venetoclax. For the conditions without Bcl-2 inhibitor (indicated with a ‘-’), the green bars indicate the use of the vehicle control for venetoclax, while the blue bars indicate the use of vehicle control for BIRD-2 treatment. A ‘+’ indicates that the Bcl-2 inhibitor (BIRD-2/venetoclax) was added in this condition. SU-DHL-6 and Karpas 422 cells were treated with 15 µM BIRD-2, whereas 26 µM BIRD-2 was used to treat the RI-1 cells. SU-DHL-6 cells were treated with 500 nM venetoclax, Karpas 422 cells with 1 µM venetoclax and RI-1 cells were treated with 10 nM venetoclax. Cell death was measured 24 h after treatment. On the y-axis the percentage of living cells, which corresponds to the Annexin V-FITC- and 7-AAD-negative fraction, is shown. Data are expressed as the average ± SEM (N ≥ 3). d CI derived from cells treated with U73122/U73343 in combination with BIRD-2/venetoclax. The CI was calculated from the data shown in a, b, c. e, f, g Quantitative analysis of 4 independent experiments detecting apoptosis in Annexin V-FITC/7-AAD-stained SU-DHL-6 (e), Karpas 422 (f), and RI-1 (g) cells. Apoptotic cell death was measured as the percentage of Annexin V-FITC-positive cells. Cells were pre-treated with U73122 (1 or 2.5 µM) or U73343 (2.5 µM) for 30 min. Cell death was measured 24 h after BIRD-2 treatment. Data are shown as the ∆ apoptotic fraction, which is the difference in apoptosis between the BIRD-2-treated and the vehicle-treated fraction, and between the BIRD-2 + U73122-treated and the U73122-treated fraction, and finally between the BIRD-2 + U73343-treated and the U73343-treated fraction. Statistically significant differences were determined using an analysis of variance (ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001)