Additional file 1 of GATA-targeted compounds modulate cardiac subtype cell differentiation in dual reporter stem cell line

Additional file 1: Supplementary Figure S1. Primary screening of GATA4-targeted compounds in differentiating stem cells for the activation of a ventricular reporter gene (venGFP, Myl2-eGFP). Compounds were screened during D2-D10 window of spontaneous differentiation of mouse embryonic stem cells (mESCs). Differentiation cultures were measured on D12 of differentiation after treatment with GATA4-targeted compounds for (a) %Myl2-eGFP (ventricular, venGFP+) cells out of total cell population, (b) the mean fluorescent intensity (MFI) of venGFP. Data is presented as mean (n ≥ 2, independent experiments). Supplementary Figure S2. Compound screening for activation of ventricular reporter gene (venGFP, Myl2-eGFP). Compounds were screened during D2-D10 window of spontaneous differentiation of mouse embryonic stem cells (mESCs). Differentiation cultures were measured on D12 of differentiation after treatment with GATA4-targeted compounds for (a) %Myl2-eGFP (ventricular, venGFP+) cells out of total cell population, (b) the mean fluorescent intensity (MFI) of venGFP. Data is presented as mean ± SEM (n ≥ 3, independent experiments). Supplementary Figure S3. Compound screening for activation of atrial reporter gene (SMyHC3-TdTomato, atrRFP). Compounds were screened during D2-D10 window of spontaneous differentiation of mouse embryonic stem cells (mESCs). Differentiation cultures were measured on D12 of differentiation after treatment with GATA4-targeted compounds and measured for (a) %SMyHC3-TdTomato (atrial, atrRFP+) cells out of total cell population, (b) the mean fluorescent intensity (MFI) of atrRFP. Data is presented as mean ± SEM (n ≥ 3 (1228, n = 2), independent experiments). **P < 0.01, *P < 0.05 (T-test vs DMSO control). Supplementary Figure S4. Ratio of expression between ventricular and atrial reporter genes. Compounds were screened during D2-D10 window of spontaneous differentiation of mouse embryonic stem cells (mESCs). Differentiation cultures were measured on D12 of differentiation after treatment with GATA4-targeted compounds and measured for (a) atrial/ventricular ratio and (b) atrRFP/venGFP ratio. Data is presented as mean ± SEM (n ≥ 3 (1228, n = 2), independent experiments). **P < 0.01, *P < 0.05 (T-test vs DMSO control). Supplementary Figure S5. Characterization of chemically induced differentiation by qRTPCR. D12 cultures were collected and analyzed for markers of cell identity genes, transcription factors, progenitors, and signaling pathways. Data is presented as mean ± SEM (n = 4, independent experiments). *P < 0.05 (Wilcoxon test vs DMSO control). Supplementary Figure S6. Original whole Western blot images. Differentiating mouse embryonic stem cells (mESCs) were treated with compound 3i-1000 during D2-D10 window of differentiation and collected at D5 and D12. The cells were lysed into 1% SDS in 50 mM Tris-HCl and protein concentration was determined. a On first experiment 80 μg of protein was loaded on gel and b on second experiment 30 μg protein was loaded on gel. Membranes were immunoblotted (IB) at first with anti-GATA4 antibody and after strip wash with anti-β-actin antibody. Samples: medium only (M), DMSO (D), 3i-1000 (3 μM), 3i-1000 (5 μM). Independent experiments were repeated two times. Supplementary Figure S7. Original whole Western blot images for HEK-cells with GATA4-V5 Tet-On/Off overexpression. The cells were lysed into 4′ Laemmli buffer with 2-mercaptoethanol. From the crude cell lysate, a sample was diluted 1/5 and further 1/25 with 1′ Laemmli buffer. A 10 μl sample from each dilution was loaded on gel and immunoblotted (IB) with GATA4 or V5 antibodies. a For control, HEK-cells were transfected with rtTA, the samples were prepared similarly as for GATA4-V5 overexpression and loaded on gel with decreasing amount 1/1, 1/5, 1/25. b At the second repetition, for control, the cells with GATA4-V5 overexpression were lysed into RIPA-buffer, protein concentration was determined and 5, 10 and 16 μg samples were loaded on gel. Independent experiments were repeated two times. Supplementary Figure S8. GATA4-targeted compounds promote differentiation of ventricular cardiomyocytes in a directed differentiation assay. Compounds were added either prior to (D6-D8, cardiac progenitors) or after (D7-D9, cardiomyocytes) the onset of spontaneous beating in defined, serum-free conditions. Total fluorescence of ventricular reporter gene (venGFP, Myl2-eGFP) upon compound treatment is depicted for compounds 3i-1148, 3i-1120, 3i-1165, and 3i-1194. Data is presented as mean ± SEM (n = 4, independent experiments). Supplementary Figure S9. Protein interactome of GATA4 and NKX2-5 by BioID after CRAPomefiltering. Proteins in more than 10% (41/411) of CRAPome database experiments were discarded. Supplementary Figure S10. High sequence conservation in zinc finger domain of GATA4 among different species. Human and other mammals are unable to regenerate cardiomyocytes after birth and express an arginine (hR310, purple) at the C-terminal tail of the zinc finger. However, species with regenerative capacity, including Zebrafish (Q09JY7), Eastern newt (F2W888), Bat star (Q6XZF5), Sea cucumber (A0A2G8JQ98), African clawed frog (Q91677) and Hydra vulgaris (T2MH05) have consistent expression of lysine at the same position (green). Conserved residues responsible for C4-coordination of zinc fingers are highlighted with gray color. In the bottom row, the alignment results are represented as follows: The asterisk (*) indicates a single and fully conserved residue. A colon (:) indicates conservation between groups of strongly similar properties. A period (.) indicates conservation between groups with weakly similar properties. A number at the end of the line indicates the running number of the last amino acid of the respective sequence. Protein sequences were downloaded from UniProt Knowledgebase (UniProtKB) which contains two separate sections; UniProtKB/Swiss-Prot (SP, manually annotated) and UniProtKB/TrEMBL (TR, computationally annotated). Sequences were aligned by using Clustal Omega (European Bioinformatics Institute, EMBL-EBI). Supplementary Figure S11. BromoMAX assay shows no significant perturbation of bromodomain proteins in cell-free assays by compounds 3i-1000 and 3i-1047 at 10 μM, indicating that the acetyl-lysine like domain within the compounds do not bind directly to bromodomains. Supplementary Table S1. Taqman assays used for the characterization of embryoid bodies from chemically induced differentiation experiments. Supplementary Table S2. Structural derivatives of GATA-targeted compounds [3, 5] examined for stage-specific activation of atrial and ventricular reporter genes in differentiating pluripotent stem cells. Supplementary Table S3. Gene ontology (GO) enrichment analysis of CRAPome-filtered BioIDresults for GATA4 (320 identified proteins) determines the most abundant functional associations. Supplementary Table S4. Gene ontology (GO) enrichment analysis of CRAPome-filtered BioIDresults for NKX2-5 (359 identified proteins) determines the most abundant functional associations. Supplementary Table S5. Summary of differentially expressed genes following 30 and 120 min treatments with GATA4-targeted compound 3i-1000 versus DMSO control in neonatal rat ventricular cardiomyocytes.