Fig. 2: Mutant PKP2 modifies structure of actin filament network.

a Representative immunoblots showing the distribution of β-actin in pellet (F-actin) and supernatant (G-actin) fractions of cells encoding PKP2, or p.R735* mutant. Graph shows F-actin:G-actin ratios calculated from densitometry analysis. Data are presented as mean ± sd; n = 6. Significance was established as p < 0.05 (unpaired Student t test). b Confocal images showing phalloidin-staining of the F-actin network (blue) in mouse neonatal cardiomyocytes (MNC) expressing PKP2 or p.R735* proteins tagged with EGFP (yellow). Nuclei were stained with DAPI (gray). Magnification shows the different structure of F-actin depending on the expression of PKP2 or p.R735*. Scale bars, 20 µm and 5 µm. c Percentage of MNC with visually apparent and organized sarcomeres; n = 54 EGFP-PKP2 cells, n = 79 EGFP-pR735* cells examined over five independent experiments. d Sarcomeric periodicity in cells with organized sarcomere network in each group of cells. Red dot line defines normal sarcomeric period (1,78 µm). Boxes depict the 25^th−75^th percentile with a line showing the median. Whiskers display minimum to maximum values. n = 19, ROIs for EGFP-PKP2, n = 10 ROIs for EGFP-PKP2-p.R735*. e TTorg analysis of the captured images assessed the organization of the sarcomeres, and this is represented by the sarcomeric power metric. Calculated transverse organization level of sarcomeres is based on the calculation of the peak amplitude in the Fourier spectrum of the image at the sarcomeric frequency. Boxes depict the 25^th−75^th percentile with a line showing the median. Whiskers display minimum to maximum values (n = 19, ROIs for EGFP-PKP2, and n = 10 ROIs for EGFP-PKP2-p.R735*). f Analysis of cellular actin orientation (coherency), using the ImageJ plugin OrientationJ, was performed with an automated routine to quantitatively assess the differences among groups analyzing 20 × 20 µm ROIs from confocal images. Orientation coherency increases with the orientation anisotropy in the image (from zero for random orientations to one for unique orientations). Boxes depict the 25^th−75^th percentile with a line showing the median. Whiskers display minimum to maximum values; n = 78 ROIs for PKP2, n = 98 ROIs for PKP2-p.R735*. Statistical significance was determined by unpaired t- test with Welch’s correction (two-sided) in (c–f). Significance was established as p < 0.05. Data are presented as mean ± sd. MW molecular weight, kDa kiloDalton, % percentage, μm micrometers, Arb Units arbitrary units. Source data are provided as a source data file.