Fig. 7: Reduction of Mito-SR/ER distance with physical linkers abrogates the benefits of DIAPH1 silencing in HiPSC-CMs in H/R.

a Scheme representing the construction of FRB–FKBP12–RAPAMYCIN–Linker complex fluorescence YFP and RFP tags. b Leica SP8 confocal microscopy images at 63× magnification representing 48 h post Lipofectamine LTX transfection followed by treatment with 100 nM rapamycin for 10 min. Scale bar 25 µm. (Data reproduced from two independent experiments) c TEM images and quantification after successful transfection with mitochondria and SR/ER localization sequences with or without 100 nM rapamycin treatment to reduce Mitochondria and SR/ER to approximately 5 nm. Scale bar 0.5 µm. (Each value represents Mito-SR/ER distance in nm originating from four biologically independent samples, Kruskal–Wallis with Dunn’s pairwise comparison test was performed for p values) d Colorimetric assay to detect LDH levels in conditioned medium obtained from linker groups (n = 4–6 biologically independent samples, ANOVA with TukeyHSD pairwise comparison test was performed for p value). e MitoSOX staining to detect mitochondrial superoxide and respective quantification using NIH-ImageJ. Images were taken at 10× magnification using an EVOS epifluorescence microscope with an RFP filter. Scale bar 200 µm. (n = 6 biologically independent samples, ANOVA–TukeyHSD pairwise comparison test was performed for p value). f qPCR under linker conditions for SR/ER and mitochondrial markers. N represents biological replicates originating from at least two consecutive batches. (n = 4 biologically independent samples. Welch’s ANOVA with Games–Howell pairwise comparison test for DIAPH1, PERK, and EDEM1, Krushal–Wallis with Dunn’s pairwise comparison test for GADD34, and ANOVA with TukeyHSD pairwise comparison for BCL2 and PARKIN for p values). Data are presented as the mean ± SEM. All statistics and source data are provided as a Source Data file.