Fig. 4.

Dormant and reactivated dormant colonies have an impaired ability to re-enter the dormant state. a A total of 50,000 MCF-7 cells were incubated on fibronectin-coated 10 cm plates. The following day (day 0) DMEM/10% FCS without or with recombinant human FGF-2, IL-6, IL-8 or TGFβ1 were added to the wells to achieve a final concentration of 10 ng/ml. On day 6, cells were detached into single cell suspension by trypsin and 1000 cells/well were incubated in quadruplicate on 24 well fibronectin-coated plates. On day 7 DMEM/10% FCS without or with FGF-2 at a final concentration of 10 ng/ml was added to the cultures. On day 13, wells were stained with 0.1% crystal violet and growing and dormant colonies were counted. b MCF-7 cells were incubated on fibronectin-coated 10 cm plates. The following day (day 0) DMEM/10% FCS with FGF-2 was added to the wells to achieve a final concentration of 10 ng/ml. On day 6, DMEM/10% FCS without or with recombinant human IL-6, IL-8 or TGF-β1 was added to the wells to achieve a final concentration of 10 ng/ml. On day 12, cells were detached into single cell suspension by trypsin and 1000 cells/well were incubated in quadruplicate on 24 well fibronectin-coated plates. On day 13, DMEM/10% FCS without or with FGF-2 at a final concentration of 10 ng/ml was added to the cultures. On day 19, wells were stained with 0.1% crystal violet and growing and dormant colonies were counted. In a control experiment, MCF-7 cells were incubated on fibronectin-coated 24-well plates at 1000 cells/well. DMEM/10% FCS without or with FGF-2 at a final concentration of 10 ng/ml was added the next day. Six days later, wells were stained with 0.1% crystal violet and growing and dormant colonies were counted. Results represent the average of quadruplicate wells. Error bars: Standard Deviation. *p < 0.05, **p < 0.01 ***p < 0.001