Plvx ires zspuro lentiviral expression vector

The vectors containing cDNA of wild-type FTO and R96Q mutant of FTO were kindly gifted by Renbin Zhao.^{40 (link)} Primers bearing Xba1 and Not1 sites were used to generate PCR fragments that were subcloned into pLVX-IRES-ZsPuro lentiviral expression vector (Clontech, Shiga, Japan). Lentiviral shRNA construct for mouse FTO gene was purchased from GENECHEN (Shanghai, China). The target sequence was: AGAACCATACTATTTGCTT. Lentivirus was produced by co-transfection of lentivirus packing plasmids with psPAX2 and pMD2.G using Jet PRIME (PolyPlus, Illkirch, France) into 293FT cells following manufacturer's instruction. Medium was changed 24 h post transfection and the medium containing virus was collected after 72 h, followed by a centrifugation at 10 000 g for 10 min. The supernatant was used to infect MPM cells in the presence of 10 μg/ml polybrene (Sigma-Aldrich, cat. H9268, Carlsbad, CA, USA), or stored at −80 °C. Selection of resistant colonies was initiated 48 h later using 3 μg/ml puromycin (Life Technology, Carlsbad, CA, USA; cat. A1113803).

Vectors containing the cDNA of WT IKKβ were obtained by amplifying mouse cDNA with Phanta® Super-Fidelity DNA Polymerase (cat. No. P511-01; Vazyme Biotech co., ltd, Nanjing, China). The IKKβ S177A mutant was obtained by amplifying the cDNA of WT IKKβ, followed by insertion into a pLVX-IRES-ZsPuro lentiviral expression vector (Clontech, Shiga, Japan). Lentivirus was produced by co-transfection of lentivirus packing plasmids with psPAX2 and pMD2.G using the Jet PRIME transfection reagents (PolyPlus) into HEK293 cells according to the manufacturer’s instructions. The medium was changed after 24 h, and the medium containing the virus was collected after another 72 h, followed by 10 min of centrifugation at 2000 × g and the supernatant was collected and stored at −80 °C. The supernatant was used to infect NIH3T3 cells with 10 μg/ml polybrene (cat. No. H9268; Sigma-Aldrich), and resistant colonies were selected after 8 h using 50 μg/ml puromycin (cat. No. 58-58-2; Sangon Biotech). miR-146a and anti-miR-146a vectors were purchased from GeneCopoeia (Guangzhou, China), and the miR-146a and anti-miR-146a lentivirus were obtained by co-transfection of lentivirus packing plasmids with psPAX2 and pMD2.G using the Jet PRIME transfection reagents (PolyPlus) into HEK293 cells according to the manufacturer’s instructions as described above.