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. 2020 Mar 20;10:5178. doi: 10.1038/s41598-020-62088-7

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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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The USP14/UCHL5 inhibitor b-AP15 and the NAE inhibitor MLN4924 are only effective at high concentrations or have a mostly cytostatic effect in GIST cells. (A) Dose-dependent effect of b-AP15 in comparison to control (DMSO) treatment on cell viability (left) and induction of apoptosis (right) of IM-sensitive (GIST882, GIST-T1) and IM-resistant (GIST48, GIST430) GIST cells as measured by luminescence-based assays (mean + /− SE). The abrupt drop of cells undergoing apoptosis in IM-resistant cells treated with 10 μM b-AP15 most likely indicates that the majority of cells were dead at this concentration and no caspase 3/7 activity (luminescence readout) was present. (B,C) Immunoblot analysis for markers of cell cycle regulation and apoptosis (B) and accumulation of mono-ubiquitinated proteins (C) in GIST cells after treatment with increasing concentrations (0.0001 μM–10 μM; 72 h) of b-AP15. Treatment with IM or SU serves as standard treatment control for IM-naïve and IM-resistant cell lines, respectively. Both were used at 1.0 μM. (D) Dose-dependent effect of MLN4924 on cell viability (left) and induction of apoptosis (right) of GIST cells as measured by luminescence-based assays (mean +/− SE). (E,F) Immunoblot analysis for markers of cell cycle regulation and apoptosis (E) and accumulation of mono-ubiquitinated proteins (F) after treatment with increasing concentrations (0.0001 μM–10 μM; 72 h) of MLN4924. Treatment with IM or SU (both 1.0 μM) serves as standard treatment controls for IM-naïve and IM-resistant cell lines, respectively. Bortezomib (BO; 0.1 µM) serves as control for proteasome inhibition. (B,E) Grouped immunoblot images are either cropped from different parts of the same gel or from a separate gel run with another aliquot of the same protein lysate.