Fig. 2.

MDAMB231 and U87 cancer cell-derived MV are capable of transforming normal fibroblasts. (A) WCL of serum-starved MDAMB231 and U87 cells, as well as lysates of the MV shed by these cells, were immunoblotted with antibodies against the MV markers actin and flotillin-2, the cytosolic-specific marker IκBα, and the activated (phospho)-EGF receptor. (B–D) Multiple sets of serum-deprived NIH 3T3 fibroblasts were incubated with serum-free medium, medium containing 2% CS, or medium supplemented with intact MV derived from either MDAMB231 or U87 cells as indicated. (B) One set of cells was lysed after being exposed to the various culturing conditions for the indicated lengths of time and then was immunoblotted with antibodies that recognize the activated and total forms of AKT and ERK. Two additional sets of fibroblasts were evaluated for their abilities to undergo serum deprivation-induced cell death (C) and to grow in low serum (2% CS) (D). For the growth assays, the culturing medium (including the MV) was replenished daily. (E) NIH 3T3 fibroblasts incubated with or without MV derived from MDAMB231 or U87 cells were subjected to anchorage-independent growth assays. The soft agar cultures were re-fed (including adding freshly prepared MV) every third day. NIH 3T3 cells expressing Cdc42 F28L were used as a positive control for these experiments. (F) Images of the resulting colonies that formed in E. The data shown in C–E represent the mean ± SD from at least three independent experiments.