File S1 - Natural Compounds' Activity against Cancer Stem-Like or Fast-Cycling Melanoma Cells

Figure S1. The influence of natural compounds used at a single concentration of 5 µM on viable cell numbers in melanoma (DMBC11 and DMBC12) and leukemia (K562) cell cultures. Viable cells were assessed by acid phosphatase activity assay (A) or by flow cytometry using an automated cell viability analyzer (B). Data are the mean ± SD of two independent experiments performed in triplicates. Figure S2. Effects of natural compounds (5 µM) on viability of melanoma cells (DMBC11 and DMBC12) and leukemia cells (K562). Changes in cell viability were assessed by PI staining and flow cytometry and they are expressed as % of vehicle control. Data are the mean ± SD of two independent experiments performed in triplicates. Figure S3. The influence of natural compounds on cell distribution in cell cycle and cell death shown as accumulation in subG1. (A) Representative histograms of DMBC12 cells treated with natural compounds at a single concentration of 5 µM for 30 h are shown. When accumulation of melanoma cells in subG1 did not exceed 40%, histograms were analyzed using ModFit software to calculate the percentages of cells in each cell cycle phase. Histograms showing cell cycle arrest were marked with green (G0/G1 phase), blue (S phase) and red (G2/M) frames, and percentages of melanoma cells accumulated in each phase are included. When accumulation of melanoma cells in subG1 exceeded 40%, FACSuit software was used and the percentages of dead cells are indicated. Results obtained for DMBC11 cells are shown in Figure 3. (B) Effects of lower concentrations for the most cytotoxic compounds or of longer exposure for compounds that were ineffective at 30 h. Figure S4. The influence of natural compounds used at a single concentration of 5 µM on the clonogenic growth of melanoma cells. Cells were incubated in drug-containing medium for 4 h and then they were grown on agar for 14 days in drug-free medium. Cell colonies were stained and counted. Anti-clonogenic activity was expressed as percentage of control treated with vehicle. At least two independent experiments were performed in duplicates. Figure S5. Dose-response curves prepared for compounds exerting anti-clonogenic and/or cytotoxic potentials. Blue curves, anti-clonogenic activity; black curves, cytotoxic activity. The graphs summarize the results of at least 3 independent experiments performed in triplicates using DMBC11 (filled square) and DMBC12 (open square) cell lines. Chemical formulas of compounds are included. Dose-response curves for more potent compounds are shown in Figure 6. Table S1. The Natural Products Set II Consisting of 120 compounds. Table contains names of all compounds and the numbers (underlined) that could be used to get detailed information about compounds from available databases. The upper numbers were given to compounds in the present study, and they appear in brackets in the text and also in some Figures. Table S2. Viability assessed after 45 h of treatment with selected compounds at indicated concentrations in six different melanoma cell lines derived from surgical specimens. Cell viability was assessed by flow cytometry after PI staining. Data expressed as % of control treated with vehicle are means ± SD of two independent experiments conducted in triplicates. Table S3. Activity profiles of natural compounds selected in this study prepared based on a literature search. Only the main biological activities of compounds are included. (PDF)