Fig. 2. Fibronectin RNAi disrupts fibrillin-1 microfibril assembly by human dermal fibroblasts. (A) RT-PCR analysis of RNAi knockdown of fibronectin in HDFs. FN1 and FN2 are knockdowns using different oligonucleotides (see Materials and Methods). FN2 scr, scrambled oligonucleotide control; Col I, collagen I; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B) Immunoblotting of fibrillin-1 [using rabbit anti-proline-rich region (PRO) pAb] and fibronectin (using FN-3E2 mAb to cellular fibronectin) on lysates and medium from knockdown and control cultures, as outlined in A, with β-actin loading controls (using mAb AC-74) for the cell lysates. (C) Representative confocal microscopy images of fibronectin assembly (using FN-3E2 mAb to cellular fibronectin) in the RNAi and control cultures, from 1-7 days. (D) Representative confocal microscopy images of microfibril assembly (using rabbit anti-proline-rich region (PRO) pAb; red) and fibronectin (using FN-3E2 mAb to cellular fibronectin; green) in control cultures, fibronectin RNAi cultures, and fibronectin RNAi cultures supplemented with cellular fibronectin (10 µg/ml), all at 5 days. Microfibril assembly was grossly disrupted in the fibronectin siRNA cells, and partially rescued by cellular fibronectin. Scale bars: 50 µm. (E) Enlarged images from D. All experiments were repeated at least three times.