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. 2019 Sep 16;9:13327. doi: 10.1038/s41598-019-49774-x

Figure 2.

Figure 2

(A) Upper panel: Quantification of the formation of the mammospheres made from the suspension of MCF-7CD44+/CD24− single cells untreated and treated with the investigated compounds. The cells were treated with increasing concentrations of the investigated compounds and equitoxic concentrations corresponding to their respective IC30,72h values (determined using CellTiter-Glo® 3D Cell Viability Assay after 72 h of treatment, cisplatin 10.2 μM, salinomycin 0.63 μM, 1 0.52 μM, 2 0.15 μM). Cells were incubated for 5 days, and mammospheres with a diameter above 40 μm were counted. Results are expressed as the mean number of mammospheres formed from 1000 viable single cells. Dashed lines represent the range of the SD determined for the control (untreated) cells. Error bars indicate SDs from three independent experiments each made in octuplicate. Cell clumps were not included in the analysis. Statistical analysis was calculated with non-parametric students´ t-test; the symbols (*) and (**) denotes a significant difference (p < 0.05) from the untreated control. Bottom panel: Representative bright-field images of the mammospheres formed from MCF-7CD44+/CD24− untreated or after 5 days of the treatment with salinomycin and complexes 1 and 2 at their respective IC30,72h (see the legend to the upper panel of this Fig. 2A). I, Untreated control; II, cisplatin; III salinomycin; IV, 1; V, 2. Scale bars represent 200 μm. (B) Growth inhibitory effects of the investigated compounds in the preformed MCF-7CD44+/CD24− mammospheres. The preformed mammospheres (the mean diameter was~200 μm) were treated with the increasing concentrations (in the range of 0.1–50 µM as indicated in Fig. 2A, upper panel) of the investigated compounds and also with their equitoxic concentrations corresponding to IC50 values determined for the treatment lasting 72 h shown in Table 1 (columns marked in Fig. 2A, upper panel as IC50) for 72 h, visualized every 24 h of the treatment and analyzed for their sphere mass. Upper panel: Quantification of the mammosphere formation. Bottom panel: Representative bright-field images of the mammospheres obtained after 72 h of the treatment at their IC50 values. I, control; II, cisplatin; III, salinomycin, IV, complex 1; V, complex 2. Cells were seeded in quadruplicate. Scale bars represent 200 μm. Error bars indicate SDs from three independent experiments each made in octuplicate. Statistical analysis was calculated with non-parametric students´ t-test; the symbol (*) denotes a significant difference (p < 0.05) from the untreated control.