Fig. 6: Insulin stimulates Py230 cell proliferation, motility, migration, and tumor spheroid formation.

a Cell counts of Py230 breast cancer cells after treatment with 0, 5, or 10 nM insulin for 24 h (n = 6 replicates of treatment per group, ***p < 0.01 by one-way ANOVA with Tukey’s multiple comparisons test). Vehicle treated cells are colored cyan, cells treated with 5 nM insulin in blue, and cells treated with 10 nM insulin in magenta. b Representative image of Py230 cell migration in transwell chambers following 0, 5, or 10 nM insulin treatment for 24 h. c Quantitation of cells that have migrated through transwell membrane (n = 4 replicates of treatment per group performed twice, **p < 0.01 by one-way ANOVA with Tukey’s multiple comparisons test). d Representative images of Py230 cell migration into a scratch wound after 0, 5, or 10 nM insulin treatment for 16 h. Black bars indicate distance between sides of scratch. e Quantitation of scratch width (n = 10, n represents number of scratches from triplicate experiment performed twice, ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test). f Representative image of tumor spheroids formed by Py230 cells in response to 2.5 or 5 nM insulin for 7 days. g–j Quantification of total number of tumor spheroids, number of round spheroids, number of irregular spheroids, and area of irregular spheroids per well (n = 3 replicates of treatment per group performed twice, *p < 0.05, **p < 0.01 by one-way ANOVA with Tukey’s multiple comparisons test). Vehicle treated cells are colored cyan, cells treated with 2.5 nM insulin in green, and cells treated with 5 nM insulin in blue. Data are presented as mean ± SEM. Asterisks show statistical significance as indicated.