Figure 3.

A: Western blot analysis of caveolin-1 expression in the nontumorigenic human fibroblast cell line IMR-90 and in the human fibrosarcoma cell line HT-1080 (top). Lane 1: IMR-90 cells express high levels of the 24-kd [α]- and the 21-kd [β]-isoform of caveolin-1. Lane 2: Caveolin-1 expression is strongly down-regulated in HT-1080 fibrosarcoma cells. Lane 3: Inhibition of the MAP kinase pathway by treatment of HT-1080 cells with the MEK inhibitor PD 98059 (50 μmol/L) for 48 hours potently up-regulates the expression of the 24-kd [α]-isoform of caveolin-1. Lane 4: Treatment with the inhibitor of DNA methylation, 5-aza-2′-deoxycytidine (1 μmol/L) for 48 hours, only marginally up-regulates caveolin-1. To ensure equal loading amounts, the blots were stripped and re probed with an antibody to actin (bottom). B: Colony formation assay demonstrating growth suppression of HT-1080 fibrosarcoma cells by expression of caveolin-1. Two independent experiments are shown. HT-1080 cells were transfected with either the caveolin-1 expression plasmid or the empty pLNHX vector as a control. Cells were selected in medium containing 850 μg/ml of G418 for 10 days, fixed, stained, and the colonies were counted. C: Western blot analysis of caveolin-1 expression of expanded G418-resistant colonies transfected with the caveolin-1 plasmid (top). No stable overexpression is observed in HT-1080 clones with the caveolin-1 plasmid. Lane 1: IMR-90-positive control; lane 2, parental HT-1080 cells; lanes 3–7, individual G418-resistant HT-1080 clones transfected with the caveolin-1 plasmid; lane 8, neo control (pLNHX). Bottom: actin control.