Fig. 6.

Inhibition of miR-193b reduces metformin-mediated inhibition of mammosphere formation. a BT-549 cells stably expressing miR-193b-Zip and miR-Scr-Zip were seeded in non-adherent conditions in Mammocult medium for 24 h prior to dose–response treatment (0–5 mM) of metformin. Second passage mammospheres (2P) counted from six biological replicates (n = 6), ***P < 0.0001 relative to untreated controls. b Images of the mammospheres were taken using Nikon phase-contrast microscope with either 5 mM metformin or vehicle control treatment. c (top) Generated mammospheres were then collected and stained with CD24/CD44 then processed by flow cytometry. Flow cytometric profiles are shown for 5 mM metformin or vehicle control treated cells. (bottom) Bar graph is representative of triplicate repetitions of experiment for gated population of cells expressing CD24^−/low and CD44^+/high. d Mammospheres were processed for Aldefluor expression using ALDEFLUOR assay. Flow cytometric profile of Aldefluor^pos cells were quantified by calculating the percentage of fluorescent cells compared with a DEAB staining reaction. Bar is representative of triplicates +/− SE. Data are representative of three independent experiments. Error bars are SEM. **P < 0.01, ***P < 0.001, two-way ANOVA with Dunnett’s multiple comparisons test