Fig. 7: Increased exosomal cargo release in human iPSC-derived cardiomyocytes upon Wnt activation.
a Confocal immunofluorescence images showing nuclear translocation and accumulation of the transcriptionally active P-Ser675-CTNNB1 in TNNT2-positive iPSC-derived cardiomyocytes upon CHIR99021 (CHIR) treatment validating WNT/β-catenin signaling induction versus DMSO control-treated cells. Western blot showing increased (b) TSG101 levels and (c) Ubiquitinated protein abundance in exosomal preparations from CHIR-treated iPSC-derived cardiomyocytes versus control (DMSO), (TGFβ1 was included as additional stress control in b) upon equal amounts of loaded proteins as visualized by the stain free gel image (biological replicates n = 3 (b) and n = 2 (c) technical replicates). In b exosome preparation from β-catΔex3 and control hearts were included for direct comparison. d Western blot and corresponding quantification showing increased TSG101 levels in exosomal preparations from CHIR-treated iPSC-derived cardiomyocytes versus control (DMSO) and a partial rescue upon concomitant treatment with Iso-Quercetin (Iso QC), blocking Wnt transcriptional activity. e Western blot showing transcriptionally active CTNNB1 and CRYAB expression in cell lysates from CHIR, Iso QC and CHIR/Iso QC treated iPSC-derived cardiomyocytes. f Confocal immunofluorescence images showing cytosolic accumulation of CRYAB in CHIR-treated iPSC-derived cardiomyocytes. Hoechst33342 was used for nucleus visualization and corresponding semiquantification is depicted, which is showing the intensity of CRYAB staining normalized to the total TNNT2 intensity (n = 6, technical replicates, biological duplicates). Scale bar = 20 µm. Data are shown as mean ± SEM; unpaired Student’s t test.
