Fig 2: Analysis of iPSC cardiomyocytes contractility. A Beating rate in cardiomyocytes overexpressing DYRK1A in control conditions (left) and after exposure to daunorubicin (DAU) (middle). Right: relative DYRK1A fold expression in transfected cells. Each point represents individual measurements in a region of interest (ROI) (n = 45–55 ROIs per condition). Each bar represents the mean ± SD of three measurements performed in triplicates for each condition. *P < 0.05, ns not significant, Student’s t test. B Representative fluorograms showing beating patterns from controls and treated cells. C Representative live-confocal microscopy images of iPSC cardiomyocytes showing changes in Fluo-4 AM intensity (green) over time. Scale bar: 50 µm

Fig 3: Impact of DYRK1A expression and drug treatments on the viability of iPSC cardiomyocytes. A Cellular viability after incubations with daunorubicin (DAU) and epigallocatechin gallate (EGCG). B Viability in controls and DYRK1A-overexpressing cells. Mean ± SD from two determinations performed in triplicates. ***P < 0.001, **P < 0.01, ns not significant, One-way ANOVA (Tukey’s test) and Student’s t test

Fig 4: SRSF6 phosphorylation in iPSC cardiomyocytes overexpressing DYRK1A. A Evaluation of the anti-phosphoepitope SR antibody in total protein extracts from AC16 cardiomyocytes treated with calf intestine alkaline phosphatase (CIAP). B Detection of phospho-SRSF6 and SRSF6 in iPSC cardiomyocytes transfected with empty vector (EV), SRSF6-HA expression construct (SRSF6) or co-transfected with DYRK1A and SRSF6-HA (SRSF6 + DYRK1A). β-Tubulin was assayed as loading control. Lower panel: densitometric analysis. Upper right panel: relative DYRK1A mRNA fold expression. (A.U.: arbitrary units). C Detection of phosphorylated SRSF6 in immunoprecipitated samples from iPSC cardiomyocytes. Right panel: densitometric analysis. Horizontal bars show the mean ± SD from determinations performed in quintuplicate. D Dot-blot analysis of phosphorylated SRSF6 in immunoprecipitated samples. Right panel: densitometric analysis. Horizontal bars show the mean ± SD from determinations performed in quintuplicate. ***P < 0.001, *P < 0.05, Student’s t test

Fig 5: The absence of CDKL5 alters the DYRK1A pathway. (A) Representative WB showing the expression level of CDKL5, DYRK1A, total Tau and Tau phosphorylated on T212 in WT/Cdkl5-KO neurons or KO neurons treated with 1 μM EGCG or 0.1 μM harmine from DIV7-DIV10. GAPDH was used as internal standard. (B) Quantification of DYRK1A expression, the ratio between phosphorylated and total Tau (pT212/tTau) and total Tau levels in Cdkl5-KO neurons treated as indicated. Data are represented as fold change (mean ± SEM) compared to the untreated WT neurons indicated by the dotted horisontal line. Statistical analysis: 1-WAY ANOVA followed by Dunnett's post-hoc test (*p < .05). n = 3–10 mice/group from at least 3 different preparations.