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. 2021 Jan 27;11:2391. doi: 10.1038/s41598-021-81351-z

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Salicylic diamines inhibit basal respiration and maximal respiratory capacity to a different extent in miPSCs, hiPSCs and miPSC-CMs. (a, c–e) Basal respiration and OCR response to FCCP-induced (1 µM) protonophoric uncoupling in undifferentiated αPIG-AT25 miPSCs after 16 h of SM treatment (a), in undifferentiated NP0040 hiPSCs after 24 h of treatment (c) and in purified miPSC-CMs after 48 h of SM treatment (d) as well as after 72 h of recovery following treatment (e). Data are shown as relative values compared to 0.05% DMSO-treated control cells and are presented as mean ± SD of n = 3 measurements from one experiment in (a and c), and n = 6 measurements pooled from two independent experiments in d and e. Each measurement was performed with 7 replicates per group. See also Supplementary Fig. S5. (b) Immunoblot analysis. miPSCs and miPSC-CMs were treated with 10 µM SM6 or vehicle (0.05% DMSO) for 8 h and then fractionated into cytoplasmic (Cy), mitochondrial (Mi) and nuclear (Nu) fractions. Levels of p53, cytosolic marker α-tubulin (α-TUB), and mitochondrial marker voltage-dependent anion channel (VDAC) were determined in each fraction and compared with those from whole cell lysates (WCL) by immunoblotting. C1 and C2 indicate control WCLs prepared from, respectively, human HEK293 and HT29 cells in the panel for iPSCs, and HEK293 and COS9 cells in the panel for iPSC-CMs. They served as positive controls for p53. Molecular weight of human p53 is slightly higher than that of murine p53 which explains different positions of this protein in murine iPSC and iPSC-CM (*) and human control samples in C1 and C2 lanes (◄). Full-length immunoblots of all analyses are shown in Supplementary Figs. S8, S9 and S10. KDa kilodalton, M protein marker.