Fig. 1: Mitochondria show significant fragmentation in skin fibroblasts from RAB7^V162M patients.

Human skin fibroblasts from two healthy controls and two CMT2B patients were cultured as described in Materials and Methods. Fibroblasts were incubated with Mito Tracker and mitochondrial images were captured by live cell imaging. a, b: representative images of mitochondria from two healthy controls; c, d: representative images of mitochondria from two CMT2B patients. To better illustrate the mitochondria morphology, a small inset from each image (white box) was magnified and presented. The images were analyzed and quantitated using the Mitochondria Analyzer Plugin in Fiji (ImageJ). The measurements for mean area (e), mean perimeter (f), aspect ratio (g), form factor (h), the number of branch junctions/mitochondrion (i), the number of branches/mitochondrion (j), total branch length (k) and mean branch length (l) are presented. Results are shown as mean ± SEM. The numbers of mitochondria were analyzed are: n = 1369 from 14 images (~30 cells) for healthy control group and n = 2075 from 11 images (~25 cells) for CMT2B patient group. In m, Western blot analysis was performed on cellular lysates from healthy control fibroblasts (n = 2) and CMT2B fibroblasts (n = 3) using specific antibodies against pDrp-1Ser616 and total Drp-1. The relative levels of pDrp-1Ser616 were quantitated and normalized against total Drp-1 (n). Densitometric analysis of immunoblot was performed using Image Lab software (BIO-RAD). Values are the mean ± SEM of three different and independent experiments. *p < 0.05, **p < 0.01, ***p < 0.0001. Significance analysis was carried out using Prism. Statistical significances were calculated by unpaired t-test. All p values are shown in the graphs.