Arsenic trioxide inhibits Hedgehog and Notch pathway target gene expression. A) Quantitative PCR analysis revealed that ATO significantly inhibited transcript levels of the Hedgehog pathway target gene PTCH1b at 5 μM in all 3 glioblastoma neurosphere lines compared to vehicle treatment, and in 2 lines after 2.5 μM ATO. ATO also significantly inhibited another Hedgehog pathway target N-Myc in 2 lines at 5 μM, in all 3 glioblastoma neurosphere lines at 2.5 μM, and in 2 lines at 1 μM. GLI2, a 3rd Hedgehog pathway target, was significantly inhibited in all 3 lines at 5 μM ATO, in 1 line at 2.5 μM ATO, and in 2 lines at 1 μM ATO. B) ATO also significantly inhibited expression of the Notch target HES1 as compared to vehicle treatment in HSR-GBM1 and 040622 cells, but not in 040821. HES5 and HEY1 transcript levels were also reduced in all 3 glioblastoma neurosphere lines following 2.5 μM ATO treatment, but a non-significant paradoxical increase was seen at the higher 5 μM dose. C) ATO inhibited HES1 and HES5 protein expression in all 3 glioblastoma neurosphere lines at 5 μM, compared to vehicle treatment.