Fig 2: Additional in vitro studiesCompound 1 and GANT61 at various concentrations (μM) were evaluated for their ability to decrease Gli1 and Gli2 protein levels in the Hh/Gli1-dependent ASZ cells. (A–C) Membrane visualization (A) and quantification of protein levels for Gli1 (B) and Gli2 (C). Blots shown in (A) are from a single representative experiment, whereas graphs in (B) and (C) are image quantifications from two separate experiments. Comparisons between protein levels were carried out using the Student’s two-tailed t test; ∗p < 0.05, ∗∗p < 0.01. (D and E) Downregulation of Hh/GLI1 target genes in ASZ cells following 48 h (D) or 72 h (E) treatment with mock, Gli1-specific siRNA (siGli1), Gli2-specific siRNA (siGli2).

Fig 3: Structure of the GLI1/DNA complex

Fig 4: Changes in DNA sugar pucker disrupt hydrogen bonds(A–C) Key hydrogen bonds for T355, K350, and S313 in the GLI1/DNA complex (A) are disrupted in the GANT61D-GLI1/DNA (B) and 1-GLI1/DNA (C) complexes. Additional figures demonstrating the overall changes in the complex structures can be found in Figures S12−S14.

Fig 5: Principal component analysis (PCA) of GLI1/DNA complexes in the presence/absence of GANT61 and 1(A) In system 1 (GLI1/DNA), the Z and B forms of DNA move toward each other, whereas the Gli1 consensus DNA sequence (blue box) moves away, which results in a bending of the DNA. (B) In the presence of either GANT61D or 1, the Z and B forms of double-stranded DNA move parallel to each other, while the GLI1 consensus DNA sequence (blue box) twists around its axis.