Cells were immersed in a solution of 2.5% glutaraldehyde in PHEM buffer (1×, pH 7.4) overnight at 4°C, rinsed in PHEM buffer, and postfixed in a 0.5% osmic acid for 2 hours in the dark and at room temperature. After two rinses in PHEM buffer, cells were dehydrated in a graded series of ethanol solutions (30 to 100%) and were embedded in EMbed 812 using an Automated Microwave Tissue Processor for Electronic Microscopy (Leica). Ultrathin sections (70 nm; Leica-Reichert Ultracut E) were collected at different levels of each block. These sections were stained with uranyl acetate and lead citrate before examination in a Tecnai F20 TEM at 200 kV in the CoMET Montpellier Rio Imaging (MRI) facilities, Institute for Neurosciences of Montpellier, France. To evaluate the frequency of contact between the mitochondria and the ER in the different fibroblasts, we randomly took 20 images of cytoplasmic area from 20 different cells in each sample with a JEOL 1400 TEM at ×15,000 magnification. In each image, we counted the number of mitochondria (403 in control cells and 649 in patient cells for a total of 1052 mitochondria) and calculated the proportion of mitochondria in close contact with ER (<30 nm). Moreover, the perimeter of each mitochondria and the proportion of the mitochondrial surface closely associated with ER were calculated. For experiments with knockdown of NCS1, 257 mitochondria were analyzed for siScr and 239 mitochondria were analyzed for siRNA NCS1.

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