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Supplementary Figure 3

Supplementary Figure 1: Metabolites plasma concentrations (MPR and oxo-4-HPR) determined in mice after single oral treatment of 4 doses of Bio-nFeR: 10, 50, 100 and 200 mg/Kg. The analysis was performed after 0, 24, 48 h. Supplementary Figure 2: Evaluation of the 50% inhibitory concentration (IC50) of BionFeR and NCI-FeR formulation was done on a representative lung cancer stem cell line (LCSC). The cells were exposed to the indicated drug doses (0-30 M) and cell viability was evaluated by CellTiter-Glo after 72 hours. Supplementary Figure 3: A) Sphingomyelin-Ceramide (SM-CER) and SphingomyelinDihydro-Ceramide (SM-DH-CER) content in lung (LC), melanoma (MEL) and colon (CRC) tumors treated with Bio-nFeR at single 100-150 mg/Kg administration were quantified through Ultra High Performance Liquid Chromatography (UHPLC), normalized to control and plotted as fold change. B) Quantification of Glucosyl-Ceramide (GLC-CER) and Glucosyl-Dihydro-Ceramide (GLC-DH-CER) in the same samples as in A, normalized to control and plotted as fold change. Supplementary Figure 4: Cancer stem cell marker expression in CSC-culture conditions and in CSC-derived xenografts. A) Flow cytometry analysis of Aldefluor in (left panels) and CD44v6 (middle and right panels) in the indicated CSC and xenograft samples. B) Immunoblot analysis of CSC antigens c-Kit and SOX-2 in the indicated control (-) or BionFeR treated (+) melanoma xenografts. -actin blot was used for equal loading control. Supplementary Figure 5: Growth curves of lung CSC-derived xenografts in control mice or mice treated with Bio-nFeR or NCI-FeR at 100 mg/kg dose for the indicated times. Mean± standard error is shown. *P<0.05