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. 2019 Jun 12;9:8553. doi: 10.1038/s41598-019-44908-7

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© The Author(s) 2019

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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Effect of mutations of VH and caspase-3 on AIDA response. One clone of each type was grown for 48 hours, with and without doxycycline and analyzed for induction of apoptosis by flow cytometry with Annexin-V (FITC) and 7-AAD (Panels A,B). Panel (A) shows the effect of the VH mutations in the single vectors, whilst panel B shows the effect of VH and CASP3 mutations is to reduce induction of apoptosis in VH-proCASP3 + VL-proCASP3 expressing cells. The viability (panel C) and caspase-3 activity status (panel D) of these clones was judged after 48 hours using the ApoToxGlo (Promega) combined assay in the presence (red bars) or absence (blue bars) of doxycycline. Viability measurements were averaged and converted to a percentage value for comparison between clone types. Caspase-3 activation was determined by the increase in luminescence following cleavage of a reporter peptide. Data were converted to fold activation value, with no doxycycline readings given a nominal value of 1 to allow comparison between clone types. Error bars on graphs in C and D represent standard deviation from the mean.