Figure 2

Biological responses of 4T1 engineered cells. (A) Wound healing assay. Cells were seeded onto 6-well plates till confluence. After 24 h, we generated wounds in the confluent monolayers. Incubation in serum-free medium was continued for additional 30 h in the presence or absence of 0.1, 1.0, and 10 nM insulin. Randomly chosen wound fields were photographed under a microscope at time = 0 and after 6 h, and 24 h. The histogram represents the mean of the migration index calculated as follows: wound area after the indicated period/initial wound area. Experiments were performed in triplicates and data calculated as means ± SE. Statistical significance was analyzed using the Student’s t test. (B) Cell invasion. Cells were seeded onto Matrigel-coated chambers in the presence or absence of insulin (0.1, 1.0, and 10 nM) for 18 h. Cells migrated to the lower compartment and adhering to the bottom surface of the membrane were quantified. The number of migrated cells after insulin exposure was expressed as the percentage of migrated cells over 4T1/IR-A cells migrated in the absence of insulin (basal). Data are presented as means ± SE of three independent experiments. (C) Cell viability. 4T1/EV, 4T1/IR-A, or 4T1/IR-B cells were incubated with or without insulin at doses of 0.1, 1.0, and 10 nM and evaluated by MTT assay. Values represent the mean ± SEM of three independent experiments performed in triplicate. (D) Cell proliferation. The same cells as in (C) were incubated with or without insulin (10 nM) and cell number measured by trypan blue exclusion assay. Values are means ± SE of three independent experiments. (E) Colony formation. Cells were seeded in soft agar, as described in Methods, and grown in 5% charcoal-stripped serum for 3 weeks. Colonies were then stimulated or not with insulin at the concentrations of 1.0 and 10 nM, stained with MTT, and photographed. The first histogram represents the number (mean ± SE) of total colonies from three independent experiments, each in duplicate wells. The second histogram represents the bigger colonies (mean ± SE) counted. (ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).