Velcade

Full-length MICALL2 was PCR-amplified from the pCMV-SPORT6-MICAL-L2 plasmid (YouBio, Hunan, China) and cloned into the pCMV-C-HA or pEGFP-N1 vector (Beyotime, Nantong, China) as previously described (Min et al., 2019 (link)). For plasmid construction, the cDNA of the c-Myc gene was amplified by PCR from NCI-H1299 cells and inserted into the pCMV-N-Flag vector (Beyotime). All the constructs were verified by sequencing. When the cells had reached ~80% confluence, they were transfected with the relevant plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
siRNAs targeting MICAL-L2 were purchased from China GenePharma Co., and contained the following sequences: siMICAL-L2 #1, 5′-GGUUCCCACAAAGAGUAUATT-3′; siMICAL-L2 #2, 5′-CUCGACGUUUGUGACAACUTT-3′; siMICAL-L2 #3, 5′-CCAAGUUCCGCUUGUCCAATT-3′. The cells were transfected with MICAL-L2 siRNA or control siRNA using Lipofectamine 2000 at 80% confluence.
The transfected cells were treated with cycloheximide (CHX) (Sigma-Aldrich, Saint Louis, MO, USA), MG-132 (Selleck Chemicals, Houston, TX. USA), Velcade (Selleck Chemicals), acadesine (AICAR; Selleck Chemicals), or chloroquine diphosphate (Chlq; MedChemExpress, Monmouth, Junction, NJ, USA) at the indicated time points.