Hemagglutinin ha ubiquitin

The FBXO11 and Snail coding sequences were inserted into pcDNA3.1 (+) vector, designated pc‐FBXO11 and pc‐Snail, respectively. The siRNA targeting FBXO11 and scramble NC were obtained from GenePharma (Shanghai, China). The pc‐FBXO11 (1 μg·mL⁻¹), pc‐Snail (1 μg·mL⁻¹) and corresponding empty vector (1 μg·mL⁻¹) were transfected into Huh7 cells using Lipofectamine 3000 (Thermo Fisher Scientific). The FBXO11 siRNA or NC was transfected into L02 cells using Lipofectamine 3000 (Thermo Fisher Scientific). The Snail‐FLAG and hemagglutinin (HA)‐ubiquitin vectors were purchased from Addgene (San Diego, CA, USA). The transfection efficiency was confirmed using western blot analysis.

MSCV-IRES-GFP, MSCV-ICN1-IRES-GFP and MSCV-ShRNA-ICN1-IRES-GFP were kindly provided by Professor Hudan Liu, Medical Research Institute, Wuhan University, Wuhan, China. pLVX-ShRNA1, pLVX-ShRNA2 and pLVX-IRES-ZsGreen vectors were purchased from Clontech Laboratories. Hemagglutinin (HA)-Ubiquitin, pMD2.G, psPAX2 and pCL-Eco were purchased from Addgene. pCMV-Blank, pCMV-N-MycTag and pCMV-N-Flag were purchased from Beyotime Biotechnology. A modified pLVX-ShRNA2-mCherry vector in which the ZsGreen fluorescent marker was replaced by an mCherry fluorescent marker was made using HiFi DNA Assembly Master Mix (NEB, Cat No. E2621L). ShSIRT1 and ShScramble were cloned into pLVX-ShRNA2-mCherry. ShScramble, ShMYC and Shp27-human were cloned into pLVX-ShRNA2. The SIRT1 coding region was cloned into pCMV-N-MycTag, pCMV-N-Flag and pLVX-IRES-ZsGreen. Mutant SIRT1-H363Y vector was generated using Q5® site-directed mutagenesis kit (NEB, Cat No. E0554). Sanger sequencing of the SIRT1-H363Y plasmid verified the presence of the desired mutation (Fig. S3a). CDK2, SKP2, MYC and mCherry were cloned into pCMV-Blank. CDK2 and p27 were cloned into pCMV-N-Flag. Shp27-mouse and ShRen were cloned into MSCV-ShRNA-ICN1-IRES-GFP.