Fig. 2: HSCHARME characterization in hiPSC-derived human CM.

A Schematic representation of the applied CM differentiation protocol from hiPSCs. B RT-qPCR amplification of readout mRNAs and HSCHARME from hiPSCs-derived CM at specific time points (D0-D20). Data were normalized to ATP5O mRNA and represent relative expression means (2^^-ΔCt) ± SEM of 3 biological replicates. Statistical test: one-sample, two-tailed Student’s t-test on LogFC values against the null hypothesis of zero represented by the control sample (highest expression time point). Significant p-values are indicated. C Schematic representation of the genome editing strategy design followed to obtain ∆P hiPSCs using CRISPR/Cas9 technology. HR template = Homologous Recombination template. D Schematic representation of HSCHARME isoforms as reconstructed by genome assembly. Arrows represent the position of primers for RT-qPCR. E RT-qPCR amplification of pCHARME and mCHARME from hiPSC-derived CM at specific time points (D0-D20). Data were normalized to ATP5O mRNA and represent relative expression means (2^^-ΔCt) ± SEM of 3 biological experiments. Statistical test: one-sample, two-tailed Student’s t-test on LogFC values against the null hypothesis of zero represented by the control sample (highest expression time point). Significant p-values are indicated. F Quantification of the subcellular distribution of pCHARME and mCHARME from D10 CM. The histogram shows the RT-qPCR quantification of the relative % of RNA abundance in cytoplasmic versus nuclear compartments and represents mean ± SEM of 3 biological experiments. GAPDH and pre-GAPDH RNAs were used, respectively, as cytoplasmic and nuclear controls. G Representative RNA-FISH staining for pCHARME (red) combined with TNNT2 immunofluorescence (green) in WT and ∆P D20 CM. The images include the maximun 2D projection of full-size confocal caption, digital magnification of highlighted region (white squares) and pCHARME signal distribution inside the nuclei (dotted lines). A plot displaying the Average Fluorescence Intensity (AFI) of pCHARME and DAPI signals of a single focal plane is also shown. Overlapping lines indicate colocalization along the horizontal distance of the selection (white square). The images are representative of 3 biological replicates with similar results.