Figure 5

Electron microscopy and western blot of Collagen VI. a) Electron microscopy of collagen VI secreted by fibroblasts from the UCMD patient (A, C) and controls (B, D). Collagen VI microfibrils from the patient (A, arrows) appear randomly deposited in the extracellular matrix, and unable to interconnect and develop regular webs with respect to normal fibroblasts (B). A ring structure constituted by a single microfilament is shown in the inset in panel A. Collagen VI fibrils from the patient are disorganized, and poor alignment was detected in the globular domains of parallel microfibrils (C, arrows). In contrast, fibrils from normal fibroblasts show the typical regular beaded organization and parallel association of globular domains (D, arrows). Scale bars: 400 nm in A-B, and 100 nm in C-D. b) Western blot of collagen VI in cell layer and medium of the UCMD patient and a healthy donor. Samples derived from skin fibroblasts of the patient (UCMD) and a healthy donor (CTRL), were separated by SDS-PAGE onto 3-8% polyacrylamide under reducing conditions. The molecular weight markers (kDa) are indicated on the left. The arrows point to the three truncated α1 (VI) products, while the asterisk refers to a non-specific band. The loading control in the cell layer samples was performed using a GAPDH antibody. The expected migration of collagen VI chains are indicated on the right.