Fig. 3. MVP facilitates GLI1 nuclear localization and stabilization.

A SW1353 and HCS2/8 cells were transiently transfected with scrambled siRNA and siMVP for 48 h, the knockdown efficiency was determined using western blot. B Western blot analysis of GLI1 distribution after transient knockdown of MVP followed by nuclear-cytosolic protein isolation in HCS2/8. HSP70 and Lamin B were used as cytoplasmic and nuclei markers, respectively. The relative expression ratio of GLI1 was shown below the blot. C Immunofluorescence analysis of GLI1 distribution in HCS2/8 after transient transfection with siMVP for 48 h, non-specific target scramble siRNA was used as the control. D Real-time RT-PCR results showing GLI1 target gene expression after MVP knockdown in HCS2/8. Error bars represent SD (n = 6). E Stable shMVP HCS2/8 cell clones were constructed, and chromatin was immunoprecipitated with the antibody of GLI1 and isotope control IgG. Eluted DNA was PCR-amplified using primers encompassing the GLI binding site of the PTCH1-promoter or the GAPDH coding region. F SW1353 and HCS2/8 cells were treated with cycloheximide (CHX, 50 μg/ml) for the indicated times, and cell lysates were analyzed with western blot with the indicated antibody. β-actin was used as the loading control, quantification results are shown in the right panel. G SW1353 and HCS2/8 cells were pre-treated with DMSO or MG132 (10 μM) for 8 h, followed by parallel co-treatment with siMVP for 24 h. Cells were lysed and analyzed by western blot with indicated antibodies. All data are presented as the mean ± SD (*p < 0.05, **p < 0.01,***p < 0.001, by Student’s t-test).