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Fig. 1. Cholesterol depletion and caveolin knockdown inhibit the effects of uPAR stimulation on matrix assembly. (A) Monolayers of A1-F cells were treated with increasing doses of MβCD for 30 minutes to deplete cholesterol. After washing, cholesterol was reintroduced into some of the cells (+) by incubating cholesterol-depleted cells with cholesterol (1 mM):MβCD (10 mM). Cells were then incubated with either 50 µM of the uPAR ligand, P25, or the control peptide, S25. Cell layers receiving no peptide served as additional controls (labeled as `C'). 125I-fibronectin (125I-FN) was added to the medium for 6 hours. Cell layers were extracted in 1% deoxycholate, and soluble and insoluble material was separated by centrifugation. The amount of 125I-FN incorporated into the detergent-insoluble matrix was determined by 
scintillation. Representative data is shown; n=3. The error bars represent the s.e.m. of triplicates. All data were normalized against the control value, which was set at 1. (B) Cells were transfected using oligofectamine with siRNA directed against caveolin-1 or a control non-targeting siRNA for 72 hours. Cells were lysed in gel sample buffer under reducing conditions and lysates were electrophoresed, immunoblotted and analyzed for the expression of caveolin. Blots were then stripped and re-probed for total Erk2 as a loading control. Representative data is shown; n=4. Caveolin knockdown was evident at 72 hours and persisted through to 96 hours. (C) The blots shown in B were scanned and the data was normalized to Erk2. Values from control siRNA cells were set at 1. Error bars represent s.e.m.; n=4. (D) Cells shown in B were treated with 50 µM of P25 or the control peptide, S25, and the 125I–70-kDa-fibronectin-fragment was added to the medium and incubated with the cells for 1 hour. Cells were then rinsed in PBS and solubilized in 1 N NaOH. The amount of 125I–70-kDa that was associated with the cell layers was determined by scintillation. (E) Cells shown in B were incubated with P25 or S25 peptides in the presence of 125I-FN for 6 hours. Cells were then rinsed in PBS and solubilized in 1 N NaOH. The amount of 125I-FN that was associated with the cell layers was determined by scintillation. Data in D and E are presented as fold increase over control, in which cells incubated in the absence of any peptide (labeled as `C') serve as control. Bars represent the standard error of triplicate samples. Asterisk (*) indicates that P25 stimulation of matrix assembly in siRNA-knockdown cells is statistically different from control cells receiving non-targeting siRNA (t-test, P<0.05).
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