Lentivirus overexpressing mouse IKKβ, METTL3, METTL14, KAT2A, KAT2B, SIRT1, SIRT2, SIRT6, SIRT7, Spi2a, FTO or DERPINA3F, and human IKKβ, METTL14, KAT2B, SOCS1 or SERPINA3 were generated via cloning the coding region of mouse Ikkβ [NM_001159774.1], Mettl3 [NM_019721.2], Mettl14 [NM_201638.2], Kat2a [NM_020004.5], Kat2b [NM_020005.4], Sirt1 [NM_019812.3], Sirt2 [NM_022432.4], Sirt6 [NM_181586.4], Sirt7 [NM_153056.3], Spi2a [NM_009251.2], Fto [NM_011936.2], Serpina3f [NM_001168294.1] cDNA or human METTL14 [NM_020961.4], SOCS1 [NM_003745.1], IKKβ [NM_001556.3], KAT2B [NM_003884.5], SERPINA3 [NM_001085.5] cDNA into pLV[Exp]-Neo-EF1A lentiviral vector (VectorBuilder). Next, using these vectors as templates, an HA tag was added in the N-terminal of Kat2b, Spi2a, SERPINA3; a Flag tag was added in the N-terminal of Kat2a; a His tag was added in the N-terminal of Mettl14, Ikkβ, and IKKβ. Site-specific mutation of METTL14 and METTL3 was constructed by a QuickChange Site-Directed Mutagenesis Kit (Agilent) based on the manufacturer’s protocols. Packaging plasmids and lenti-vector were co-transfected into HEK293T cells. 48 h after transfection, lentivirus was collected from the cell culture medium and added into macrophages with 4 µg/ml polybrenes. The fragment bearing m6A site in the cDNA of Spi2a was subcloned to the downstream of Luc gene of luciferase reporter vector pGL3-Promoter (Promega, Cat#: 200517) to generate the pGL3-Spi2a plasmid. pGL3-Spi2a-Mut was generated by mutating the m6A motif sequence 5’GAACC3’ to 5’GATCC3’ in pGL3-Spi2a plasmid using the Mutagenesis Kit above. All of the mutations were confirmed by DNA sequencing. Primers were listed in Supplementary Table 1.
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