Cytotoxicity driven by falcarindiol (FAD) on glioblastoma cells. (A) Western blot analysis of Bax, γH2AX, and cleaved caspase 3 in U373 glioblastoma cells after treatment of dimethyl sulfoxide (DMSO), FAD (10 μM, 40 μM), and TMZ (200 μM) for 3 days. β Actin was used as a loading control. The relative band intensities of each proteins are shown below the bands. The intensities of vehicle treated lane were arbitrarily set as 1. (B) The mRNA expression of Bax, Bad, and p21 after treatment of DMSO, FAD (40 μM), and TMZ (200 μM) for 3 days in glioblastoma cells, as measured by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. The mRNA level of DMSO-treated cells was set to 1. Representative results from multiple experiments are shown.

Downregulation of the stemness of glioblastoma stem-like cells by FAD treatment (A) Cell morphology of U373 glioblastoma cells in two different culture conditions. For the enrichment of glioblastoma stem-like cells, cells were maintained under N2 culture media supplemented with 20 ng/mL EGF and bFGF (N2+GF). Spheres are maintained with N2 media including 1% fetal bovine serum (FBS) for differentiation induction (N2 + 1% serum). (B) Western blot analysis of Nestin and GFAP in undifferentiated or differentiated glioblastoma cells. β Actin was used as a loading control. The relative band intensities of Nestin and GFAP proteins are shown below the bands. The intensities of N2+GF lane were arbitrarily set as 1. (C) Western blot analysis of Nestin and GFAP in undifferentiated or differentiated glioblastoma cells after treatment of DMSO, FAD (10 μM, 40 μM), and TMZ (200 μM) for 3 days. β Actin was used as a loading control. The relative band intensities of Nestin are shown below the bands. The intensities of vehicle treated lane were arbitrarily set as 1. (D and E) Immunofluorescence analysis of Nestin (D) and GFAP (E) expression in DMSO or FAD treated cells. Nuclear DAPI (4′,6-diamidino-2-phenylindole) staining is in blue. These are representative results of counting 10 random fields of view in each group.

Disruption of the cellular maintenance by FAD in normal neural stem cells (NSC)s. (A) Sphere-forming capacity of NSCs in the absence (DMSO) or at various concentrations of FAD. Scale bar = 100 μm. (B) Western blot analysis of phosphor Histone 3 (Ser 10) and cleaved caspase 3 in normal NSCs after treatment of DMSO, FAD, and other single compounds (10 μM) for 3 days. β Actin was used as a loading control. The relative band intensities of each proteins are shown below the bands. The intensities of vehicle treated lane were arbitrarily set as 1. (C) Western blot analysis of Nestin, phosphor ERK, total ERK, phosphor Histone 3 (Ser 10), Cyclin D in DMSO or FAD-treated cells. Total ERK was used as a loading control for phosphor ERK. In other cases, β Actin was used as a loading control. The relative band intensities of each proteins are shown below the bands. The intensities of vehicle treated lane were arbitrarily set as 1. (D,E) Immunofluorescence analysis of phosphor Histone 3 at Ser 10 (shown in red) (D) and Nestin (shown in green) (E) expression in DMSO or FAD treated cells. Nuclear DAPI (4′,6-diamidino-2-phenylindole) staining is in blue. Cells were maintained under N2+GF media. These are representative results of counting 10 random fields of view in each group.

Differentiation induction of NSCs by FAD. (A) Immunofluorescence analysis of GFAP (green) and βIII-tubulin (red) expression in NSCs kept under differentiation media for 3 days. Nuclear DAPI (4′,6-diamidino-2-phenylindole) staining is in blue. These are representative results of counting 3 random fields of view. (B) Western blot analysis of GFAP and βIII-tubulin in DMSO or FAD-treated cells. β Actin was used as a loading control. The relative band intensities of each proteins are shown below the bands. The intensities of vehicle treated lane were arbitrarily set as 1. (C) Immunofluorescence analysis of GFAP (green) and βIII-tubulin (red) expression in NSCs under differentiation condition after treatment of DMSO or FAD for 3 days. (D) The quantification of GFAP- and βIII-tubulin-positive cell proportions in DMSO or FAD treated cells. These are representative results of counting 10 random fields of view in each group.

Downregulation of FoxO-Notch axis in NSCs by FAD. (A) Csl-induced luciferase reporter activity was measured in DMSO or FAD treated NSCs. Statistical differences were considered significant when ** p < 0.01, and *** p < 0.001. (B) The mRNA expression of Hes1 and Hes5 after treatment of DMSO or FAD in NSCs, as measured by real-time RT-PCR analysis. The mRNA level of DMSO-treated cells was set to 1. Representative results from multiple experiments are shown. Statistical significance was determined by unpaired t-test. * p < 0.05, and ** p < 0.01. (C) Western blot analysis of FoxO1 and FoxO3 in NSCs after treatment of DMSO or FAD for 24 h or 48 h. β Actin was used as a loading control. The relative band intensities of each proteins are shown below the bands. The intensities of vehicle treated lane were arbitrarily set as 1. (D) The mRNA expression of FoxO1 and FoxO3 after treatment of DMSO or FAD in NSCs, as measured by real-time RT-PCR analysis. The mRNA level of DMSO-treated cells was set to 1. Representative results from multiple experiments are shown.