Fig 1: MVP promotes GLI1 expression by activating the mTOR/S6K1 signaling cascade.A Western blot analysis of principle proteins involved in IHH, mTOR, and MAPK signaling pathway in control and CS cells with MVP knock-down. Densitometry analysis was performed to quantify the relative phosphorylation levels of p70S6K1, AKT, mTOR, and ERK based on the blots. B Western blot results showing the expression of indicated proteins in SW1353 cells over-expressing GLI1 and OUMS27 cells with GLI1 knockdown. C SW1353 and HCS2/8 were treated with dual PI3K/mTOR inhibitor PF-04691502(PF) for 24 h, cell lysates were subjected to western blot analysis with the indicated antibody. β-actin served as the loading control and the relative expression of GLI1 and phosphorylated p70S6K1 and mTOR are shown below the blots. D Real-time RT-PCR results showing the expression of genes related to the IHH pathway after treatment with PF-04691502 for 24 h.Error bars represent SD (n = 6). E Western blot analysis showing expression of proteins associated with IHH and mTOR pathway in HCS2/8 cells over-expressing MVP transfected with shp70S6K1 in the presence or absence of 10 ng/ml EGF. All data are presented as the mean ± SD (*p < 0.05, **p < 0.01,***p < 0.001, by Student’s t-test).
Fig 2: GLI1 forms complex with MVP and mTOR through the region of SUFU binding sites.A Silver staining results of IP with GLI1 antibody in SW1353 cell lysates. Normal IgG was used as the negative control. B SW1353 and HCS2/8 cells were lysed and subjected to IP-western blot assay to determine the interaction between endogenous GLI1 and MVP. C Immunofluorescence analysis of GLI1 and MVP distribution in CS cell lines. DAPI was used to stain nuclei. D The interaction between MVP and mTOR pathway components was examined using IP with MVP antibody. E HEK-293T cells were transiently transfected with empty vector or Flag-GLI1 together with GFP-MVP, cells were lysed and subjected to IP-western blot assay 48 h after transfection to examine the interaction of GLI1, MVP, mTOR, and p70S6K1. F HEK-293T cells were transiently transfected with MVP-GFP, GLI1-trunc1-Flag (SUFU binding sites), GLI1-trunc2-Flag (zinc-finger DNA-binding domain), and GLI1-trunc3-Flag (transcription activation domain). Cells were harvested 48 h after transfection followed by IP-western blot assay. G HEK-293T cells were transiently transfected with MVP-GFP, GLI1-trunc1-Flag, and SUFU-V5. Cells were harvested 48 h after transfection followed by IP-western blot assay.
Fig 3: Simultaneous inhibition of MVP and GLI1 strongly inhibits the growth of CS.A Cell viability of SW1353 after treatment with GANT61 or/and shMVP. Error bars represent SD (n = 6). B In vivo combination therapy for subcutaneously inoculated tumors using shMVP or/and GANT61 (40 mg/kg). In total, 5 × 106 HCS2/8 cells or HCS2/8 clones stably transfected with shMVP were subcutaneously injected into the right flank of nude mice, tumor cells were allowed to grow for 7 days before GANT61 or the solvent (corn oil) were administered. GANT61 was subcutaneously injected and the tumor volumes were measured and calculated every 3 days. Error bars represent SD (n = 4, the GANT61 single-agent group n = 3). C Images of tumors from the indicated groups and weights of resected tumors. Error bars represent SD (n = 4, the GANT61 single-agent group n = 3). D Representative IHC images of Ki67 and P-p70S6K1 in resected tumors. E Schematic representation of MVP-mediated GLI1 stabilization, nuclear localization and activation.
Fig 4: GLI1, MVP and P-p70S6K1 expression are positively correlated in human CS tissues.A Western blot results showing expression of GLI1, MVP, P-p70S6K1, and P-AKT in tissues from normal articular cartilage, osteochondroma, and conventional CS. Densitometry was performed to quantify the relative expression of these proteins as shown in the right box plot. B Western blot results showing expression of GLI1, MVP, P-p70S6K1, and P-AKT in four CS cell lines. C Representative images of positive or negative IHC staining results of GLI1, MVP, and P-p70S6K1. D Statistical analysis of IHC staining results from 71 human conventional CS tissues. E Statistical analysis of positive IHC staining results for MVP, GLI1, and P-p70S6K1 in five normal cartilage and 71 conventional CS tissues. F Statistical analysis showing correlation between GLI1 and MVP in IHC staining results of 71 human conventional CS tissues. G Statistical analysis for the correlation between P-p70S6K1 and GLI1 based on IHC staining results of 71 human conventional CS tissues. H Statistical analysis for the correlation between P-p70S6K1 and MVP based on IHC staining results of 71 human conventional CS tissues.
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