Additional file 1 of Small molecule-mediated rapid maturation of human induced pluripotent stem cell-derived cardiomyocytes
Creators: name: Chirico, Nino
name: Kessler, Elise L.
name: Maas, Renée G. C.
name: Fang, Juntao
name: Qin, Jiabin
name: Dokter, Inge
name: Daniels, Mark
name: Šarić, Tomo
name: Neef, Klaus
name: Buikema, Jan-Willem
name: Lei, Zhiyong
name: Doevendans, Pieter A.
name: Sluijter, Joost P. G.
name: van Mil, Alain
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Cancer
subject: Computational Biology

description: Additional file 1: Table S1. Overview of culture Media used during this study. Table S2. Primer list used for qPCR experiments. Table S3. Antibodies list used for western blot, flow cytometry and fluorescent Immunohistochemistry experiments. Fig. S2. Cell line-dependent small molecules dose titration. iPSC line-dependent dose–response effect on CMs at day 27 of differentiation (Fig. 2). For further experiments, the cell clone-dependent optimal concentrations (UKKi036-C: AA 2 and 1 μM, GW: 250 and 100 nM; and T3: 200, and 100 nM; UKKi032-C: AA 10 and 5 μM, GW: 1000 and 500 nM; and T3: 400, and 200 nM; UKKi037-C AA 10 and 5 μM, GW: 1000 and 500 nM; and T3 800 and 400 nM – Fig. S2) were used. Fig. S3. AA and GW treatment enhances levels of mitochondrial key enzymes expression. a) OGDH CTRL versus AA, GW, and T3, respectively: 1.110 (0.6000 – 1.800) versus 1.919 (1.285–2.375; p = 0.0078) versus 2.608 (2.035–2.963. n.s.) versus 2.287 (1.520–2.770; p =0.0156); b) NDUFV3 CTRL versus AA, GW, and T3, respectively: 1.037 (0.7100–1.400;) versus 1.738 (1,553 – 1.933, p =0,0078), 3.750 (1.738 – 4.390; n.s.). and 2.251 (1.950 – 2.510; p=0.0156); c) COX3 CTRL versus AA. GW. and T3, respectively: 1.187 (0.4200–1.830) versus 1.166 (0.8725–1.433; p = 0.0234) versus 2.860 (1.918–3.753; p = n.s.) versus 2.203 (1.760–2.880; p = 0.0156); d) COX5 CTRL versus AA. GW. and T3, respectively: 1.053 (0.7800–1.540) versus 2.471 (1.950–2.943; p = 0.0078) versus 9.758 (6.838–13.52; p = n.s.) versus 2.873 (2.460–3.320; p = 0.0156). Fig. S4. Experiments were performed using day 27 iPSC-CMs line UKKi036-C. (a) Raw extracellular acidification rate (ECAR) of iPSC-CMs cultured in the Seahorse medium with L-glutamine. Addition of ETO and 2DG block the FAO and glucose-dependent glycolysis, respectively. (b) Raw oxygen consumption rate (OCR) in cells at day 27 of differentiation after sequential administration of ETO and 2DG. Table S4. Relative DNA content per single nucleus measured on three independent experiments with iPSC lines: UKKi036-C UKKi032-C, and UKKi037-C combined n = 6–12. Fig. S5. Cell nuclei count per well measured by Hoechst staining and 20X magnification imaging using the Evos microscope and ImageJ show no significant changes in number of nuclei present. Fig. S6. Full-length blots of connexin-43 (left) and CaV1.2 (right) with marker and molecular weight of protein of interest in red. Western blots were performed on pooled proteins from all three cell lines. Equal amount of protein was loaded in each lane. Ponceau staining is shown underneath the western blot panel as a loading control.