Fig 1: IMP inhibits GLI1 transcription through STAT3. (A) Heatmap of GLI2 and STAT3 target genes in 143B cells treated with or without IMP from GEO database (GSE180321). (B) Western blot analysis of GLI1, P-STAT3 and STAT3 expressions in Light II cells treated with SAG (100 nM) with or without IMP (10 μM, 20 μM) treatment for 24 h. The β-actin was shown as a loading control. (C) GLI-luciferase activity in shSUFU-Light II cells with or without STAT3 overexpression (100 ng) in the presence or absence of IMP (20 μM). (D) Molecular docking of the binding model between IMP and STAT3 (PDB: 1BG1). All experiments were repeated at least three times. Statistical significance was calculated using Student t test, #P>0.05, *P <0.05.
Fig 2: IMP inhibits Hh signal at the level of GLI. (A) Western blot analysis of SUFU expression in wild-type (shCtrl) and SUFU-knockdown (shSUFU) Light II cells. The GAPDH was shown as a loading control. (B) GLI-luciferase reporter activity in shSUFU Light II cells treated with IMP (10 μM, 20 μM) or GDC (1μM) for 36 h. (C) Western blot analysis of GLI1 expression in shSUFU-Light II cells treated with IMP (10 μM, 20 μM) or GDC (1μM) for 24 h. The GAPDH was shown as a loading control. (D and E) Dose-response inhibition of GLI-luciferase activity by IMP in Light II cells with overexpression of GLI1-Flag (D) or GLI2-Myc (E). (F) Western blot analysis of Flag expression in Light II cells with overexpression of GLI1-Flag. The β-actin was shown as a loading control. All experiments were repeated at least three times. Statistical significance was calculated using Student t test, #P>0.05, *P <0.05.
Fig 3: OLA1 reduced the GEM sensitivity of PDAC by the SHH/HHIP axis to activate Hh pathway(A) Effects of silencing OLA1 on Hh pathway genes.(B) Effects of OLA1 knockdown and (C) overexpression on Hh pathway proteins.(D) Expression of OLA1 and HHIP in OLA1-induced subcutaneous tumor model.(E) Effects of GEM and GDC-0449 on the viability of OLA1-overexpressing cells. Subsequent analyses included tumor photos (F) and measurements of tumor weight (G) in mice with subcutaneous tumor xenografts. Additionally, animal tumor imaging was performed to assess tumor size and weight (H).(I and J) Immunohistochemical (IHC) staining was conducted to evaluate the expression of OLA1, Ki67, SHH, Gli1, and HHIP in tumor tissues. Scale bars of 100 μm (left) and 50 μm (right) were included for reference. n.s., not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Fig 4: Schematic illustration of the function and mechanism of IMP in overcoming the resistance of SMO inhibitors. IMP inhibited GLI1 transcription by acting at its promoter via STAT3, thereby circumventing various resistance mechanisms of clinical available anti-Hh drugs, including SMO mutations, loss of SUFU and GLI2 amplifications.
Fig 5: IMP impairs the growth of Shh-medulloblastoma in vivo. (A) The tumor volume of DAOY allografts over the time course of treatments with vehicle control (Con) or IMP (50 mg/kg) once daily (n=5). (B) qRT-PCR analysis of GLI1 expression in treated DAOY tumors (n=5). (C) Western blot analysis of GLI1, P-STAT3, and STAT3 expressions of treated DAOY tumor tissues. (D) Representative images of H&E staining of treated DAOY tumor tissues (n=5). (E) Representative images of Ki67 staining of treated DAOY tumor tissues (n=5). (F) Quantification of Ki67 positive cells in tumor tissues from E. All error bars represent mean ± s.d. Statistical significance was calculated using Student t-test or two-way ANOVA, *P <0.05, ***P <0.001.
from Cell Signaling Technology for Hedgehog Signaling Antibody Sampler Kit