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. 2016 Nov 24;8(1):1007–1022. doi: 10.18632/oncotarget.13537

Figure 3. Inhibitory effects of benztropine mesylate on BCSC properties in vitro.

Figure 3

A-B. Mammosphere formation assays: Number of mammospheres (diameter > 50 μm) from 1,000 MDA-MB-231 or 4T1-luc2 cells which were treated with different concentrations of deptropine citrate, benztropine mesylate, salinomycin or paclitaxel for 6 days was counted. C-D. Self-renewal assays: Adherent cells were pretreated with or without the compounds at indicated concentrations for 4 days and mammosphere formation was evaluated in sequential sphere generations without any treatment. Data are expressed as mean±SD (n=6). FACS analysis of the expression of CSC markers (ALDH+ and CD44+/CD24−) in MDA-MB-231 spheres with or without benztropine mesylate treatment. MDA-MB-231 spheres were treated with benztropine mesylate (1, 5 or 10 μM), salinomycin (2 μM), paclitaxel (10 nM) or DMSO for 6 days. Single cell suspensions were used for FACS analysis. Representative data for ALDH+ E. and CD44+/CD24− G. populations in benztropine mesylate-treated MDA-MB-231 spheres show a reduction compared with DMSO-treated cells. DEAB was used to inhibit the reaction of ALDH with the ALDEFLUOR reagent, providing a negative control. The proportions of ALDH+ F. and CD44+/CD24− H. cells are shown as mean±SD. Experiments (n=3) were conducted in triplicate. **p<0.01, ***p<0.001 compared with DMSO control (one-way ANOVA).