IkappaB Kinase beta

Protein–ligand docking was conducted for eight targeted proteins under nine systems-TLR4, p38-α MAPK (ATP-binding site), p38-α MAPK (non-ATP-binding site), ERK1, ERK2, JNK1, JNK2, JNK3 and IκB kinase β using crystallized structures (retrieved from RCSB Protein Data Bank), listed as follows: 2Z65^{44 (link)}, 3ZSH^{33 (link)}, 1KV2^{45 (link)}, 4QTB^{46 (link)}, 1PME^{47 (link)}, 2NO3^{48 (link)}, 3NPC^{49 (link)}, 4W4Y^{50 (link)}, 4KIK^{51 (link)}, respectively. In addition to protein preparation, the 3-dimensional structures of 1 and 2 were manually constructed using the Gaussian 09 program and optimized using the HF/6-31d basis set implemented in the program⁵² . For docking studies, the Gold docking program was used, and protocols were as follows: (1) the ligand binding site was defined as 6 Å for sphere docking and (2) ChemScore was used for the scoring function. Binding between proteins and compounds was visualized in Accelrys Discovery Studio 2.5 (Accelrys Inc.).
For all systems, docking was validated using aforementioned protocols to redock the corresponding crystallized ligand (Table S2), followed by aligning redocked pose with its original conformation to compare its similarity termed root-mean-squared-deviation (RMSD) of structure coordinates. For TLR4, since no crystallized small molecule inhibitors targeting the TLR-MD2 protein–protein interface were available, docking was performed by rational validation. Briefly, we manually docked a previously studied compound (ZINC25778142)^{34 (link)} and the docked pose was then observed its orientation and intermolecular interactions with the key reported residues including D209, S211, and D234. Our docking protocols were validated for all systems (Supplementary Information: Figure S61).

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.

Cells or tissues were lysed in RIPA buffer containing 5% 2-mercaptoethanol and Protease Inhibitor Cocktail on ice for 5 min, and denatured at 95 °C for 10 min. Protein concentration of each sample was measured by the BCA Protein Assay Kit. Proteins in lysates were separated by SDS–PAGE and then transferred onto 0.45 μm polyvinylidene fluoride membranes. The membranes were blocked in 2% BSA for 1 h at room temperature, followed by overnight incubation with primary antibodies (1:1000 dilution) in cold room. On day 2, membranes were washed by TBST buffer and incubated with anti-mouse or anti-rabbit IgG-horseradish peroxidase(HRP)-conjugated secondary antibodies (1:2500 dilution). After washing, bands were detected by ECL solution and visualized by x-ray films in dark room. Details of antibodies are: Anti-METTL3 (Aviva Systems Biology, Cat#: ARP39390_T100), Anti-SERPINA3 (Abcam, Cat#: ab129194), Anti-KAT2B (Abcam, Cat#: ab12188), Anti-KAT2A (Abcam, Cat#: ab217876), Anti-Phospho(Tyr) (Abcam, Cat#: ab179530), Anti-p-IKKα/β (Cell Signaling, Cat#: 2697), Anti-IKKγ (Cell Signaling, Cat#: 2685 Anti-IKKβ (Cell Signaling, Cat#: 8943), Anti-HA (Cell Signaling, Cat#: 3724), Anti-IκBα (Cell Signaling, Cat#: 4814), Anti-Phospho(Ser/Thr) (Cell Signaling, Cat#: 9631), Anti-Acetylated-Lysine (Cell Signaling, Cat#: 9441), Anti-SIRT1 (ProteinTech, Cat#: 13161-1-AP), Anti-SIRT2 (ProteinTech, Cat#: 19655-1-AP), Anti-SIRT6 (ProteinTech, Cat#: 13572-1-AP), Anti-SIRT7 (ProteinTech, Cat#: 12994-1-AP), Anti-His (ProteinTech, Cat#: 66005-1-Ig), Anti-Flag (ProteinTech, Cat#: 20543-1-AP), Anti-mouse IgG-HRP (Santa Cruz Biotechnology, Cat#: sc-516102), Anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Cat#: sc-2357), Anti-Spi2a (Santa Cruz Biotechnology, Cat#: sc-57013), Anti-IKKα/β (Santa Cruz Biotechnology, Cat#: sc-7607), Anti-β-actin (Santa Cruz Biotechnology, Cat#: sc-47778), Anti-METTL14 (Sigma-Aldrich, Cat#: HPA038002), Anti-EIF3A (Cell Signaling, Cat#: 2538). Dilution for all of the primary antibodies is 1:1000.