Ha gsk3 β k85a

EGFP-LC3 construct was obtained from Addgene (plasmid #24920). HA-GSK3-β-K85A, a kinase dead GSK3-β mutant, was obtained from Addgene (plasmid #14755). Lipofectamine 2000 was used for transfection of HA-GSK3-β-K85A as per the manufacture’s protocol. pEGFP-LC3 was transfected into cells using Lipofectamine 2000 and visualized with fluorescence microscopy 48 hours post- transfection. siRNA specific to DRAM1(siGENOME 55332), along with a corresponding non-targeting negative control was obtained from Thermo Fisher. siRNA pools were transfected at 100 nM using Lipofectamine 2000 and assayed for knockdown at 48 hours post-transfection; or subjected to drug treatment 24 hr post transfection.

FLAG-UBR5 (#37188), HA-GSK3β (#14753), HA-GSK3β-S9A (#14754), and HA-GSK3β-K85A expression plasmids were from Addgene (USA). FLAG-AMPKα1 (CH805185), FLAG-SKP2 (CH896343), and FLAG-AKT1 (CH846646) were purchase from Vigenebio (Shangdong, China). SIRT7 and GSK3β deletion mutants were constructed into the p3× FLAG-CMV10 vector (Sigma). GST-tagged SIRT7 or SIRT7-S259A/Thr263A/Thr255A was constructed by cloning into pGEX-4T-3 (GE Healthcare). The other SIRT7 mutants were generated by the site-directed mutagenesis using the KOD-Plus-Neo DNA polymerase (TOYOBO). All plasmids were confirmed by DNA sequencing (Genewiz). Details of the primers used for the generation of these plasmids or other experiments are shown in Supplementary Table 2.