Additional file 1 of Canonical Wnt signaling activation by chimeric antigen receptors for efficient cardiac differentiation from mouse embryonic stem cells
Creators: name: Sogo, Takahiro
name: Nakao, Shu
name: Tsukamoto, Tasuku
name: Ueyama, Tomoe
name: Harada, Yukihiro
name: Ihara, Dai
name: Ishida, Tomoaki
name: Nakahara, Masato
name: Hasegawa, Koji
name: Akagi, Yuka
name: Kida, Yasuyuki S.
name: Nakagawa, Osamu
name: Nagamune, Teruyuki
name: Kawahara, Masahiro
name: Kawamura, Teruhisa
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biotechnology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Fig. S1. TCF/β-catenin-dependent transcriptional activities of mouse ES cells stably expressing scLRP6S and scFZD8S. TCF/β-catenin-dependent luciferase activities in ES/mock and ES/scLRP6S/scFZD8S following stimulation with BSA-FL are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The values of mock-transduced cells treated with vehicle were set to 1. The data represent the mean of triplicates and standard deviation from one representative experiment. Fig. S2. TCF/β-catenin-dependent transcriptional activities in mouse ES cells stably expressing scLRP6S and scFZD8L. TCF/β-catenin-dependent luciferase activities in ES/mock and ES/scLRP6S/scFZD8L cells following stimulation with CHIR99021 (a WNT activator) or BSA-FL at indicated concentrations are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The values of mock-transduced cells treated with vehicle were set to 1. The data represent the mean and standard deviation. Fig. S3. TCF/β-catenin-dependent transcriptional activities of mouse embryonic fibroblasts (MEFs) expressing scLRP6S and scFZD8L. TCF/β-catenin-dependent luciferase activities in MEFs expressing mock or scLRP6S/scFZD8L following stimulation with BSA-FL at the indicated concentrations are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The values of mock-transduced cells treated with vehicle were set to 1. The data represent the mean and standard deviation from triplicate measurements. Fig. S4. Western blotting for non-canonical Wnt activation in scLRP6S and scFZD8L-expressing cells. Protein expression levels of Ror2 and β-actin in mock- or scLRP6S/scFZD8L-expressing ES cells (A) or NIH3T3 cells (B) treated with Wnt5a, Wnt3a or BSA-FL are shown. Fig. S5. BSA-FL diffuses into EBs. Representative fluorescence microscopy images in EB sections from ES/mock and ES/scLRP6S/scFZD8L cells treated with vehicle or BSA-FL. Nuclei are counterstained with DAPI showing BSA-FL deposition possibly in the plasma membrane. The right panels are enlarged images of the boxed area of the BSA-FL-treated EBs. Scale bars = 50 μm. Fig. S6. Incidence of spontaneously beating EBs derived from three independent ES cell lines expressing scLRP6S and scFZD8L. The incidence of spontaneously beating EBs stimulated by 0.1 μg/ml BSA-FL, 50 ng/ml Wnt3a, or vehicle for 24 h from day 2 of differentiation is shown. Time course of the incidence of spontaneously beating EBs differentiated from ES/scLRP6S/scFZD8L stimulated by 0.1 μg/ml BSA-FL, 50 ng/ml Wnt3a, or vehicle for 24 h from day 2 after EB formation. Fig. S7. BSA-FL enhances cardiac differentiation of EBs expressing scLRP6S and scFZD8L. Representative flow cytometry plots of cardiac troponin T (cTnT, indicated by FITC) vs. phycoerythrin (PE) for differentiated ES/mock and ES/scLRP6S/scFZD8L treated with vehicle, Wnt3a or BSA-FL. Fig. S8. TCF/β-catenin-dependent transcriptional activities in mouse ES cells stably expressing scLRP6S and scFZD8L. TCF/β-catenin-dependent luciferase activities in ES/mock and ES/scLRP6S/scFZD8L cells following BSA-FL treatment with or without IWP-4 or XAV939, WNT inhibitors, are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The values of mock-transduced cells treated with vehicle were set to 1. The data represent the mean and standard deviation. Fig. S9. TCF/β-catenin-dependent transcriptional activities in mouse ES cells expressing single chimeric receptors. TCF/β-catenin-dependent transcriptional activities measured by luciferase reporter assay in mouse ES cells transiently transfected with plasmids encoding various chimeric LRP6 or FZD8. The values in mock-transfected cells treated with vehicle were set to 1. The data represent the mean of duplicates and standard deviation from one representative experiment. Fig. S10. Activity of BSA-FL and Wnt3a after incubation at 37 °C. TCF-dependent transcriptional activities were measured by luciferase reporter assay in mouse ES cells transiently transfected with scLRP6S and scFZD8L. The cells were stimulated with 0.1, 1, or 10 μg/ml BSA-FL or 50 ng/ml Wnt3a that were incubated at 37 °C for 0, 1, 2, 4, or 7 days. Luciferase activities of samples stimulated by each ligand with no incubation were set to 100. Fig. S11. TCF/β-catenin-dependent transcriptional activities in three independent mouse ES cell lines stably expressing scLRP6S and scFZD8L. TCF/β-catenin-dependent luciferase activities in ES/scLRP6S/scFZD8L following indicated BSA-FL treatment are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The data represent the mean and standard deviation. Table S1. List of primer sequences used for plasmid construction. Table S2. List of primer sequences used for real-time qPCR.