Fig. 5.

Inhibition of miR-193b impairs the ability of metformin to kill TNBC lines. BT-549 cells stably expressing miR-193b-Zip and miR-Scr-Zip were starved of glucose for 24 h prior to replenishing with medium containing 5 mM glucose for an additional 24 h. Cells were treated with 5 mM metformin for an additional 48 h prior to harvesting for apoptosis assays. a Cells were stained with YO-PRO1/PI stain and processed by flow cytometry. Data shown is representative of triplicate samples. Cells shown in region (A2) are indicative of dead cells, and percentage is representative of total cells harvested. Percent of apoptotic cells are quantified (right), normalized to miR-Scr control cells, error bars are SEM. **P < 0.01, two-way ANOVA with Dunnett’s multiple comparisons test. b Cells were stained with HOECHST dye, and apoptotic cells are indicated with white arrows. Percent of apoptotic cells are quantified (right), normalized to miR-Scr-Zip control cells. Error bars are SEM. ****P < 0.0001, two-way ANOVA with Dunnett’s multiple comparisons test. c Cells were processed for immunofluorescent analysis with LIVE/DEAD Viability/Cytotoxicity kit. Membrane-permeant calcein AM is cleaved by esterases in live cells resulting in bright green fluorescence, and membrane-impermeant to ethidium homodimer-1 labels nucleic acids of membrane-compromised cells resulting in a bright red color