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. 2018 Apr 23;9(5):470. doi: 10.1038/s41419-018-0502-4

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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. 1 — a Ba/F3 cells were deprived of IL-3 in the presence or absence of CT98014 (0.75 µM) and analyzed for apoptosis by Annexin V staining and flow cytometry analysis. Error bars represent SD from technical replicates. b Ba/F3 cells expressing CRISPR/Cas9 targeting Luciferase (crLUC), Puma (crPuma), or Bim (crBim) were deprived of IL-3 in presence or absence of CT98014 (0.75 µM) and analyzed for apoptosis by Annexin V staining after 18 h. Error bars represent SD from technical replicates. c Ba/F3 Puma^−/− single-cell clones and Ba/F3 expressing CRISPR/Cas9 constructs targeting Luciferase were deprived of IL-3 for 18 h and analyzed for apoptosis by Annexin V staining. Each dot represents the mean of two independent experiments analyzing an individual single-cell clone. Error bars represent 95% confidence interval from two independent experiments (n = 2). Significance was tested by one-way ANOVA with post hoc Tukey’s multiple comparison test. d Ba/F3 Bim^−/− single-cell clones and Ba/F3 expressing CRISPR/Cas9 constructs targeting Luciferase were deprived of IL-3 for 18 h and analyzed for apoptosis by Annexin V staining. Each dot represents the mean of two independent experiments analyzing an individual single-cell clone. Error bars represent the 95% confidence interval from two independent experiments (n = 2). Significance was tested by one-way ANOVA with post hoc Tukey’s multiple comparison test. ns = not significant. e Ba/F3 cells were deprived of IL-3 in presence or absence of CT98014 (0.75 µM) and harvested after the indicated time. The 0 h condition represents cells kept in medium with IL-3. RNA levels were analyzed by qRT-PCR with primers for Puma and L32 as internal reference. Data points show relative (mRNA) Puma induction compared to cells kept in medium with IL-3. Error bars represent SD from technical replicates. f Cells from a were subjected to western blotting and analyzed with the antibodies indicated.