Fig. 3.

Abnormal nuclear phenotypes are evident in patient-derived fibroblasts. a Control and patient-derived (Patient 1: III-3; Patient 2: IV-4) fibroblasts were immunostained with anti-lamin A/C and anti-protein disulfide isomerase (anti-PDI) antibodies and analyzed by confocal microscopy. Representative images are shown on the left and selected areas are enlarged. The edge of anti-PDI staining marks the nuclear envelope (NE) and a pronounced anti-lamin nuclear rim staining can be identified in the control. Quantitative analysis of anti-lamin staining intensity was performed for 2 control and 2 patient cell lines with a custom written ImageJ macro defining a 1 µm wide ring around the NE (see: Methods for further details). A summary of the relative staining intensity measurements is shown on the right (n ≥ 30; bars indicate SEM). Scale bar, 1 µm. b Cytoplasmic channels observed by DNA (Hoechst 33258) and anti-lamin A/C staining, using widefield immunofluorescence, in some of the nuclei from both patient-derived primary fibroblast cell lines. Asterisks indicate nuclei containing channels, shown in a wide field view and at a higher magnification. Channels are marked by arrowheads. The gallery at the bottom shows a collection of DNA-stained nuclei with distorted shapes containing 1–3 channels of different sizes. Such channels were only observed in patient-derived fibroblast nuclei. Quantitative analysis of channel occurrence in 2 control and 2 patient cell lines is shown in the bar chart (bottom-right; n = 150 in 4 independent experiments; bars indicate SEM). Scale bars, 20 µm