Figure 2.

NL-103 increases the acetylation levels of α-tubulin and total histones, and effectively inhibits Hh signaling by targeting Smo. (A) NIH3T3 cells were treated with varying concentrations of NL-103 or vorinostat. After 6 h, cells were harvested and the acetylation levels of α-tubulin and total histones were measured using immunoblotting. (B) Vismodegib, NL-103 and vorinostat dose-dependently inhibit Shh-N-induced firefly luciferase expression in NIH3T3-12Gli cells. Seeded cells were divided into two groups. One group was cultured in HEK293 control medium and the other was maintained in Shh-N conditioned medium. Both groups were treated with various concentrations of each compound. For cells treated with the same compound at the same concentration, reporter activity in Shh-N was normalized to that in control medium. Empirically, this calculation method can effectively filter out the nonspecific effects of compound on reporter activity. Data are the average of three independent experiments ± SD. (C) GDC-0449 and NL-103 compete for the binding of BODIPY-cyclopamine to Smo-WT-expressing HEK293T cells, but not SAHA. Nonspecific binding as defined by BODIPY-cyclopamine levels of cells treated with mock transfection. (D) Flow cytometric quantitation of specific BODIPY-cyclopamine binding to Smo-expressing cells was used to determine the affinities of Smo antagonists through binding competitions.