Fig. 3. Blocking 5β1 integrins disrupts fibrillin-1 microfibril assembly by human dermal fibroblasts. (A) FACS analysis of HDFs, confirming expression of 5β1 and vβ3 integrins. The antibodies used were mAb13 (β1 integrin), mAb16 (5 integrin), 17E6 (v integrin) and 23C6 (vβ3 integrin). (B) HDFs were incubated for 24 hours or 5 days in the presence of integrin-blocking antibodies mAb13 (β1) and mAb16 (5) or a rabbit IgG control (all at 10 µg/ml). Medium and cell lysates were immunoblotted for fibrillin-1 [using the rabbit anti-proline-rich region (PRO) pAb] or fibronectin (using FN-3E2 mAb), with β-actin (using mAb AC-74) loading controls for the cell lysates. Molecular masses are indicated (in kDa). Quantitative analysis on the media was done by densitometry as a measure of pixel density. Cell lysates were quantified using densitometry with data normalised against β-actin. All data are represented as the mean ± s.d. of two repeated experiments. There was no statistical difference between treated samples and the control when using the Student's t-test. (C) Representative confocal microscopy images of the assembly of microfibrils (using PRO pAb; red) and fibronectin (using FN-3E2 mAb to cellular fibronectin; green), in the presence or absence of mAb13 (blocks β1 integrins) and mAb16 (blocks 5 integrin), at 5 days. Cell nuclei were stained with DAPI (blue). Microfibril assembly was grossly disrupted in the antibody-treated cells. Scale bars: 50 µm. Enlarged images are also shown. All experiments were repeated at least three times.