Fig. 4. Schlafen12 reduces MDA-MB-231 Breast Cancer Stem cell stemness. A) Flow cytometry analysis of MDA-MB-231 cells 96-hours after treatment with AdSLFN12 or AdCMV showing a reduction in CD44⁺CD24^- BCSCs population and an increase in differentiated non-BCSCs population (CD44⁺CD24⁺, CD44^-CD24⁺, CD44^-CD24^-), (n=6, *p<0.05). B) SYBR Green qPCR analysis of mRNA levels of CD44 and CD24 and C) CD49F and SSEA4 in MDA-MB-231 cells 72 hours after treatment with either AdSLFN12 or AdCMV (RPLP0 used as a reference gene), data normalized to AdCMV group (CD44/24 n=6, CD49F/SSEA4 n=9, *p<0.05). D) Mammosphere formation efficiency (MFE %) of SLFN12-expressing MDA-MB-231 stable clones, (MFE% obtained by dividing the number of mammospheres over number of seeded cells then multiplied by 100), (n=18, *p<0.05). E) Mammosphere formation assay of SLFN12-expressing MDA-MB-231 stable clones represent the mean±standard error diameter of the developed mammospheres. Mammospheres <40µm were censored from the data. (n=71, *p<0.05). F) Representative images of mammospheres developed from SLFN12-expressing MDA-MB-231 stable clones or control cells after 7 days incubation in MammoCult Medium. G) Flow cytometry analysis of sorted pure CD44⁺CD24^- cell population from MDA-MB-231 cells treated with either AdSLFN12 or AdCMV for 96 hours (n=8, *p<0.05). H) Cell cycle analysis by flow cytometry and Propidium iodide (PI) labeling of the sorted CD44⁺CD24^- cells in (G) treated with either AdSLFN12 or AdCMV for 96 hours before analysis (n=8, *p<0.05). All error bars shown represent standard error of mean.