FIGURE 6:

Functional characterization of Cav1* fibroblasts. (A) Flow cytometry was used to measure the proliferation rate of human fibroblasts. Cells were incubated with 10 μM thymidine analogue EdU for 1 h, followed by fluorescence staining of cells that incorporated EdU. Cav1* fibroblasts proliferated at a twofold higher rate than control fibroblasts. (B) Collagen secretion was increased by 20% in Cav1* human fibroblasts compared with control fibroblasts. Treatment of cells with TGFβ increased collagen secretion by 30% in control fibroblasts but had minimal effect on Cav1* fibroblasts. (C) Zymography using gelatin showed that Cav1* fibroblasts secreted greater matrix metalloproteinase 9 (MMP9 or gelatinase B) than controls, based on increased gelatinolytic band at 92 kDa in Cav1* fibroblasts vs. controls. (D) Reintroduction of human WT Cav1 in Cav1^−/− fibroblasts reduced proliferation by 50%, whereas introduction of Cav1* in Cav1^−/− fibroblasts did not alter proliferation. To identify transfected cells, we cotransfected cells with Mito-DsRed plasmid (red fluorescence labeling of mitochondria). Proliferation was measured using Ki67. White arrows point to transfected cells that are not proliferating; yellow arrows point to transfected proliferating cells. Scale bar, 200 μm.