Fig 2: GLI1/IFT20 signature is correlated to primary cilium and VDAC1. A, Histograms represent the expression of the mRNA of GLI1 (left panel) and IFT20 (right panel) in RCC4 cells compared to RCC4+pVHL in Nx. B, Graphic representation of the Boyden chamber cell-based invasion assay using RCC4+pVHL and RCC4 cells. C, RCC4+pVHL cells were transfected with control siRNA (siCtl), siGLI1 (40nM), siIFT20 (40nM) and siIFT20/GLI1 (40nM+40nM). Quantitative analysis of the ciliation percentage in RCC4+pVHL cells was assessed by confocal fluorescence microscopy (n=100-300 cells). D, Triple immunofluorescence labeling and merged images with acetylated a-tubulin (Acet. a-tubulin in red), Arl13b (in green) and DAPI (in blue). E, Graphic representation of the Boyden chamber cell-based invasion assay using RCC4+pVHL cells transfected with control siRNA (siCtl), siGLI1 (40nM), siIFT20 (40nM) and siIFT20/GLI1 40nM+40nM). F, Histograms represent the expression of the mRNA of GLI1 (left panel) and IFT20 (right panel) in RCC4 cells transfected with siRNA VDAC1. Cell lysates from the same experimetn were analyzed by immunoblotting. G, Graphic representation of the Boyden chamber cell-based invasion assay using RCC4 cells transfected with siRNA VDAC1 compared to Ctl (siCtl). H, Histograms represent the expression of the mRNA of GLI1 (left panel) and IFT20 (right panel) in RCC4 cells transfected with siRNA LGMN. Cell lysates from the same experimetn were analyzed by immunoblotting. I, Graphic representation of the Boyden chamber cell-based invasion assay using RCC4 cells transfected with siRNA LGMN compared to Ctl (siCtl). The mean ±SEM is representative of three independent experiments. A * p<0.05, ** p<0.005 and *** p<0.0005 show significant differences. Experiments have been proceeded without serum.

Fig 3: A and B, Overall survival (A) and Disease free survival (B) for VDAC1 expression was calculated from Kidney renal clear cell carcinoma (KIRC) (Gepia.cancer-pku.cn) using the third quartile for the cutoff. C and D, Overall survival (C) and Disease free survival (D) for LGMN expression was calculated from KIRC. E, Tissues lysates (Normal (N), Tumoral (T)) of 19 patients were analyzed by immunoblotting for VDCA1 and LGMN. Hsp-90 was used as a loading control. Group A: high level of VDAC1 + VDAC1-ΔC and LGMN in T. Group B: low level of VDAC1 and absence of VDAC1-ΔC and LGMN. F, Expression of VDAC1-ΔC and Hsp-90 proteins was quantified in tumoral tissues (T) and VDAC1-ΔC/Hsp-90 ratio was obtained in each patient. G, Graphic representation of LGMN mRNA expression in patients from Group A compared to patients from Group B. H, HK2, RCC4+pVHL, RCC4, cell lysates were analyzed by immunoblotting for HIF-2α, HIF-1α, LGMN and VDAC1. β-tubulin and Hsp-90 were used as a loading control.

Fig 4: BSSM activates AKT/GSK3ß pathway, promotes tau hyperphosphorylation and PHF accumulation and increases the expression of active asparagine endopeptidase (AEP) and tau cleavage. (A,B) Western blotting showed higher expressions of p-AKT and the downstream p-GSK in hippocampus in BSSM treated group. (C,D) Phosphorylation of tau at S396 was higher in BSSM group than in sham and USSM group. PHF accumulation also showed significant differences between sham and the two shear stress modifier groups. (E–G) Expression of active AEP and tau N368 truncation were higher in BSSM group. (H,I) Immunofluorescent staining further reconfirmed the finding that BSSM group had more tau N368 fragments in hippocampus than the other two groups. L, left hippocampus; R, right hippocampus. *p < 0.05, **p < 0.01. Data were presented as Mean ± SD. N = 4 in each group. Scale bar, 100 µm.