Additional file 3: of Ube2v1-mediated ubiquitination and degradation of Sirt1 promotes metastasis of colorectal cancer by epigenetically suppressing autophagy
Creators: name: Shen, Tong
name: Cai, Ling-Dong
name: Liu, Yu-Hong
name: Li, Shi
name: Gan, Wen-Juan
name: Li, Xiu-Ming
name: Wang, Jing-Ru
name: Guo, Peng-Da
name: Zhou, Qun
name: Lu, Xing-Xing
name: Sun, Li-Na
name: Li, Jian-Ming
Publisher: name: figshare
Year: 2018
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Figure S1. Protein expressions of LC3-II and Beclin1 were examined by western blots when Ube2v1 was overexpressed in DLD-1 and SW480 cells under both normal medium culture condition (a) and starvation in Hank’s buffered saline solution (HBSS) (b). The cells were treated with Bafilomycin A1(BafA1), an autophagy inhibitor which blocks autophagosome–lysosome fusion. Figure S2. The effects of Ube2v1 on gene expressions of autophagy genes. A-B. mRNA levels of autophagy genes (LC3, Beclin1, ATG16L1, ATG3, ATG5, ATG7, ATG12, ATG10, ATG4a, ATG4b, ATG4c and ATG4d) were detected by qPCR analysis when Ube2v1 expression was knocked down (a) or overexpressed (b) in SW480 cells. C-D. mRNA levels of autophagy genes (LC3, Beclin1, ATG16L1, ATG3, ATG5, ATG7, ATG12, ATG10, ATG4a, ATG4b, ATG4c and ATG4d) were detected by qPCR analysis when Sirt1 expression was knocked down (c) or overexpressed (d) in SW480 cells. Figure S3. The effects of Ube2v1 on stabilization and ubiquitination of Sirt1 in CRC cells. The expression of Sirt1 was detected by western blotting in shRNA-transduced cells (a) or Ube2v1 overexpressed (b) SW480 cells treated with cyclohexamide (CHX) (100 μg/ml) for the indicated time intervals. The intensity of endogenous Sirt1 expression for each time point was quantified by densitometry. c Ubiquitination assays of exogenous Sirt1 in the lysates from SW480 cells cotransfected with GFP-Ube2v1, HA-Ub, Myc-Sirt1 or vector control. The cells were treated with or without MG132 (20 μM) before harvest and then immunoprecipitated them with anti-myc antibody. d Ubiquitination assays of exogenous Sirt1 in the lysates from SW480 cells cotransfected with HA-Ub, Myc-Sirt1 in the lysates from SW480 cells stably expressing Ube2v1 shRNA(shRNA/Ube2v1) or shRNA (shRNA/Control). Figure S4. Expressions of E-cadherin after Ube2v1 knockdown with stimulation of Autophagy inhibitor, 3-Methyladenine (3-MA) (5 mM) for 24 h. Figure S5. The effects of Ube2v1overexpression on wound-healing, migration and invasion of CRC cells. Wound healing (a), migration (b) and invasion (c) of SW480 and DLD-1 cells after Ube2v1 expression was stably overexpressed. Statistical significance was determined by using a two-tailed, unpaired student’s t-test.*p &lt; 0.05. Figure S6.The effects of rapamycin on Ube2v1 mediated in vitro migration and invasion of CRC cells. a Expressions of E-cadherin and LC3-II, were detected after Ube2v1 was overexpressed with or without rapamycin (final concentration:10 ng/ml) stimulation. B-C. In vitro migration (b) and invasion (c) assays of CRC cells after Ube2v1 overexpression with or without rapamycin (final concentration:10 ng/ml) stimulation for 36 h. Figure S7. A proposed model for the role of Ube2v1 in CRC metastasis. Ube2v1 promotes ubiquitination and degradation of Sirt1 by the help of Ubc13, reduces H4K16ac, and finally epigenetically suppresses gene expression of autophagy genes. Then, Ube2v1 promotes autophagy-dependent EMT and metastasis in CRC. (PDF 2486 kb)