Fig 1: SAE1 protein enhances Akt SUMOlyation and phosphorylation. a The expression of SAE1, SUMO1, p-AKT in 3 random chosen HGTs and their paired PBTs. b The H&E staining and the immunohistochemical staining of SAE1, SUMO1, p-AKT in 2 cases of HGTs with a high pathologic grade and their paired PBTs. c The expression of SAE1, SUMOylated Akt, p-Akt (Ser473) and global SUMOylation were measured in 2 cases of HGTs with a high pathologic grade and their paired PBTs. HGTs: human glioma tissues; PBTs: para-cancerous brain tissues; case #4–6: glioma tissues respectively with the pathologic grade IV, III and I. d The levels of SAE1 and global SUMO1 modification were detected in U87 and U251 cells. (e-f) SAE1 activates Akt SUMOylation and phosphorylation (Ser473) in glioma cells. After the plasmid SAE1 or siSAE1 were transfected into U87 or U251 cells for 48 h, cell pellets were collected to extract cellular lysates to enrich SUMOylated proteins by IP, from which the protein were eluted to detect SUMO1 modified proteins and SUMOylated AKT. Cell lysates were also detected by Western blotting respectively with anti-SUMO1, anti-SAE1, anti-AKT, anti-p-AKT and β-actin antibodies. S-AKT: the SUMOylated AKT protein. p-AKT: phosphorylated Akt. Con: pFlag empty vector as a mock control. SAE1: pFlag-SAE1. siCon: non-targeting control siRNA. siSAE1: The SAE1-specific siRNA3. IP: immunoprecipitation, IB: immunoblot, Input: same account of cell lysate to load
Fig 2: The overexpression of SAE1 is associated with metastasis and poor prognosis in patients with HCC. (A) Representative IHC photo-images and graphical representation of SAE1 protein level in paracancerous and cancerous tissues. (**** p < 0.0001) (B) Kaplan-Meier curve of overall survival according to SAE1protein level of TMU-SHH HCC cohort. (C) Representative IHC photo-images of SUMO1, SUMO2, and UBC9 protein levels in paracancerous and cancerous tissues.
Fig 3: SAE1 is a reliable diagnostic and prognostic biomarker for HCC. (A–C) Kaplan–Meier curves of overall survival, disease-specific survival and progression-free interval for HCC patients according to TCGA-LIHC database. (D) ROC analysis of SAE1 expression in non-tumor versus tumor. (E,F) Forest plot showing hazard ratio estimates and 95% confidence intervals according to TCGA studies.
Fig 4: The SUMO E1 activating subunits SAE1/SAE2 and E3 ligases PIAS1 are mainly detected in the nuclear matrix.A, a common scheme for fractionation of cellular components into cytosol, nuclear extract, and nuclear matrix and chromatin. B, HeLa cells were subjected to cellular fractionation as illustrated in (A) and Western blot analyses were performed for cytosol marker GAPDH, nuclear and nuclear matrix marker Lamin A/C, chromatin marker histone H3, and SUMO machinery components SAE1/SAE2, UBC9, and PIAS1. C, experimental scheme for preparation of chromatin-free nuclear matrix. D, HeLa cells were fractionated as illustrated in (C), and the resulting fractions were analyzed by Western blotting using antibodies as indicated. E, experimental scheme with Benzonase treatment; fractions obtained were cytosol, nuclear extract (S), nuclear matrix and chromatin (P), nuclear extract plus soluble chromatin after Benzonase digestion (S′), nuclear matrix plus remaining insoluble chromatin after Benzonase digestion (P′). F, HeLa cells were fractionated as in (E) and analyzed by Western blotting using antibodies as indicated. G, representative immunofluorescence images of HeLa cells treated with or without Benzonase as indicated and stained with antibodies specific to PIAS1, SAE1, and UBC9, respectively. DAPI staining showed that DNA was essentially depleted after Benzonase treatment. The scale bar represents 5 μm. NE, nuclear extract; NM & chro, nuclear matrix and chromatin.
Fig 5: SAE1 and UBA2 promote the proliferation and migration in NSCLC cells. A, B The CCK-8 assay of A549 and H838 cells transfected with SAE1, UBA2, or negative control plasmids. C, D Cell cycle analysis by flow cytometry of A549 and H838 cells transfected with SAE1, UBA2, or negative control plasmids. E, F Transwell migration assay results from A549 and H838 cells co-transfected with SAE1 and UBA2, or negative control plasmids. *P < 0.05, **P < 0.01, and ***P < 0.001
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