Figure 2
CPT inhibits secretion of collagenous proteins by both SSc and healthy control fibroblasts.Confluent fibroblasts in 12-well plates were incubated for 48 h in 0.1% BSA/DMEM containing ascorbic acid (50 μg/ml), and then treated for 24 h with various concentrations of CPT (none, or 10-9, 10-8, 10-7, or 10-6 mol/l). After CPT treatment, 20 μCi/ml of [3H]proline was added to the medium for an additional 24 h. Aliquots of conditioned media normalized for cell number were loaded on a 6% SDS–polyacrylamide gel. After electrophoresis, the gel was enhanced by fluorography and visualized by autoradiography. (a) Representative autoradiographs from SSc and healthy control fibroblasts. (b) Summary of quantitative analysis of the collagenous proteins expressed by SSc (left panel) and healthy control fibroblasts (right panel) after CPT treatment. Bars show means ± SEM from 10 (SSc) and 9 (healthy) independent experiments. The steady-state level of collagenous proteins in CPT-free controls ('None') was arbitrarily set at 1. (c) Representative autoradiogram of the total protein synthesis in the SSc fibroblasts after CPT treatment. Similar results (not shown) were obtained with the control cells. BSA= bovine sreum albumin; CPT = camptothecin; SSc = systemic sclerosis. *P < 0.05, **P < 0.005).