Fig 2: Involvement of Gli2 in cellular migration/invasion abilities.The (A, B) pCS2MT-Gli2ΔN, (C, D) Gli2 specific shRNA plasmids, and were transfected in cells for 24 h, then the abilities of migration and invasion were detected as described in the “Materials and Methods” section. (E-G) NGR cells were treated with specific phospho-AKT inhibitor MK2206 for 24 h. The phopho-AKTser473, phospho-GSK3βser9 and Gli2 protein were detected, and migration/invasion abilities were measured.

Fig 3: Scheme illustrates the involvement of Gli2-dependent pathway in gemcitabine resistant UC cells.The possible mechanisms identified from the in vitro results of present study were summarized in this cartoon graph. Besides the involvement of aberrant gemcitabine metabolism, gemcitabine can activate AKTSer473 phosphorylation to inactivate GSK3β kinase by phosphorylating its Ser9 residue. The inactivated GSK3β leads to stabilize Gli2 proteins to induce its downstream target genes expressions, which promotes migration/invasion abilities and resistance to gemcitabine.

Fig 4: AKT/GSK3ß pathway regulated Gli2 activation in UC cells.Cells were co-transfected with reporters and (A) pCS2MT-Gli2?N, (B) Gli2 specific shRNA plasmids, (D-F) pcAKT, Flag-GSKS9D (constitutively inactive GSK-3ß), or Flag-GSKS9A (constitutively active GSK-3ß) plasmids, respectively, and then harvested for the detection of their Gli2-luc activity and protein expression. (C) Cells were transfected with Gli-luc followed by treatment with SMO specific inhibitor, GDC0449, for 24 h. The Gli2-luc activity was measured as described in the “Materials and Methods” section.

Fig 5: 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).