Figure 5. Effect of atuveciclib on mammospheres and stem-like cancer cells from TNBC cell lines.

(A) MDA-MB-231 cells were seeded into 96-well low attachment plates into specialized spheroid formation extra cellular matrix (ECM). After 72 hours, invasion matrix was added and spheres were treated with atuveciclib at indicated concentrations. After 6 days, spheres were imaged and invaded area (μm²) was measured with ImageJ software. Data are presented as the percentages of vehicle control (VC) treated cells (upper panel). Results represent the means ± SEM of four independent experiments. Lower panels depict representative images of indicated treatment groups. ^****P < 0.0001. Scale bar represents 1000 μm. (B) MDA-MB-231 (left panel) or MDA-MB453 (right panel) cells were seeded into 96-well low attachment plates to allow formation of mammospheres (MS). Subsequently atuveciclib was added at indicated concentrations. After 8 days, mammospheres were imaged to determine cross-sectional area. Data are presented as the percentages of vehicle control (VC) treated cells. Upper panels: results represent the means ± SEM of four (MDA-MB-231) and three (MDA-MB-453) independent experiments. Lower panels depict representative images of mammospheres from the indicated treatment groups. ^***P < 0.001, ^****P < 0.0001. Scale bar represents 1000 μm. (C) MDA-MB-231 cells were seeded into 6-well plates and treated with atuveciclib at indicated concentrations. After 4 days, cells were stained with anti-CD24 and anti-CD44 antibodies and analyzed by flow cytometry. Left panel: data are presented as the percentage of CD24^low/CD44^high cells. Results represent the means ± SEM of six independent experiments. Right panels depict representative dot plots of the experiment shown in the left panel. ^****P < 0.0001. N.S. stands for non-significant. (D) MDA-MB-231 (upper left panel) or MDA-MB-453 (upper right panel) cells were seeded into 6-well plates and treated with atuveciclib at indicated concentrations. After 4 days, cells were stained with ALDEFLUOR with or without DEAB followed by flow cytometry analysis. Representative dot plots from the experiment (MDA-MB-231: left, MDA-MB-453: right) are depicted in the lower panels. Data are presented as the fold-change over vehicle control (VC) treated cells. Results represent the means ± SEM of four (MDA-MB-231) and five (MDA-MB-453) independent experiments. ^**P < 0.01; ^***P < 0.001, ^****P < 0.0001.