Hieff mut site directed mutagenesis kit

The overexpression plasmid pCMV6-Entry-EphA2-Myc-DDK was purchased from OriGene (RC205725, Origene, MD, USA). The cDNA of EphA2 was amplified and subcloned to the vector with pcDNA3.0-HA. pcDNA3.0-EphA2-HA was generated by restriction digestion of the plasmids with HindIII (R3104V, NEB, MA, USA) plus XhoI (R0146V, NEB, MA, USA), followed by ligations. The cDNA of EFNA1 was amplified from the reversed RNA and subcloned to the vector with pcDNA3.0-Flag. pcDNA3.0-EFNA1-Flag was generated by restriction digestion of the plasmids with BamHI (R3136V, NEB, MA, USA) plus XhoI (R0146V, NEB, MA, USA), followed by ligations. One-site mutant plasmids of pcDNA3.0-EphA2-S897A-HA, pcDNA3.0-EphA2-S897D-HA, pcDNA3.0-EphA2-Y588A-HA and pcDNA3.0-EphA2-K646M-HA were generated by using Hieff Mut™ Site-Directed Mutagenesis Kit (11003ES10, Yeasen, Shanghai, China). Cells were transfected using Lipofectamine 2000 (11668019, Invitrogen, Carlsbad, CA, USA) according to the instructions. Briefly, cells were seeded and grown to ~70% confluence. Related plasmid was transfected into the cells with transfection reagent for 4–6 h and then replaced with fresh culture medium.

The GenBank ID of PoRIPK1, PoRIPK3, and PoMLKL are 109624097, 109645932 and 10964593, respectively. PCR was employed to amplify the codon-optimized protein coding sequences (CDS) of PoMLKL, PoRIPK1, and PoRIPK3. Site-directed mutations of PoRIPK3 K46A, D146N, S231N/S232N and ⁴⁵⁵VQSG⁴⁵⁸−⁴⁵⁵AAAA⁴⁵⁸, and PoMLKL N105A-D106A, K231R, L293P, G318D, S360A, S361A, S360A-S361A, S360E, S361E, and S360E-S361E were performed using the Hieff Mut Site-Directed Mutagenesis Kit (Yeasen, Shanghai, China). The accuracy of the mutations was confirmed through sequencing analysis. The primers used are listed in Supplementary Table S1.

The human CaMKIIγ coding sequence (NM_172171.2) or FOXO3a coding sequence (NM_001455.3) with a 3×FLAG sequence and a kozak sequence was cloned into the vector pCDH-MSCV-MCS-EF1α-GFP+Puro (System Biosciences, Palo Alto, CA, USA) using Hieff Clone™ One Step Cloning Kit (Yeasen, Shanghai, China). Point mutation of CaMKIIγ T287A was produced using Hieff Mut™ Site-Directed Mutagenesis Kit (Yeasen, Shanghai, China) with pCDH-MSCV-CaMKIIγ+3×FLAG-EF1α-GFP+Puro as template, and the primers as follows (mutated base in lower case): forward, 5’-TCGTCA GGAGgCTGTGGAGTGTTTGCGCAAGTTCAATGCCCG-3’; revese, 5’-CACT CCACAGCCTCCTGACGATGCATCATGGATGCCACCGTG-3’.
For knockout of CaMKIIγ, we used CRISPR/Cas9 system. Two seperate sgRNAs against CaMKIIγ were designed and cloned into lentiCRISPR V2 vector (Addgene plasmid # 52961, Cambridge, MA, USA) following the the CRISPR protocol [57 (link)]. Then we evaluated them in HEK293 cells for their ability to knockdown CaMKIIγ. The sequence of the more efficacious sgRNA to target CaMKIIγ as follows: 5’-CACCGTGCTTTCTCTGTGGTCCGC-3’; 5’-AAACGC GGACCACAGAGAAAGCAC-3’.

AMPK-DN was cloned from pMIGR-AMPK-KD (Addgene, Cat #27296) into pcDNA-3xFlag plasmid. The fragment of AMPKγ2 was cloned from pGEM-PRKAG2 (#HG16130-G, SinoBiological, Beijing, China) and constructed into pLKO-puro FLAG plasmid. Mutagenesis was performed using the Hieff Mut™ Site-Directed Mutagenesis Kit (YEASEN, Shanghai, China) according to the manufacturer’s instruction. The transfection was performed using Lipofectamine 3000 reagent from Life Technologies (Carlsbad, CA) according to our described previously (Wang et al., 2021b (link)).

SIRT7 was cloned into pcDNA 3.1(+) plasmid (Addgene). The primers were designed with Primer Premier 5.0 (see in Supplementary Table 3) and cDNA was synthesized according to the manufacture of PrimeScript RT Reagent Kit (TaKaRa). PCR products were separated by electrophoresis in 1.5% agarose gel and purified using the SanPrep Column DNA Gel Extraction Kit (Sangon Biotech). Then restriction endonuclease were added into the plasmid and the purified products. Then the recombinant plasmid was constructed using the Hieff CloneTM Plus One Step Cloning Kit (Yeasen). SIRT7 mutants (S134A, S136A, S377A) were prepared using a site-directed mutagenesis protocol (Hieff Mut™ Site-Directed Mutagenesis Kit, Yeasen). All the DNA sequences of the SIRT7 constructs were assessed by a sequencing service. The construction of the HA-OGT plasmid was similar to that of SIRT7 (primers are listed in supplementary Table 3). HA-Ub, HA-Ub K63R (mutation of lysine at 63 residue to arginine), HA-Ub K48R (mutation of lysine at 48 residue to arginine), HA-Ub K63 (mutation of lysine at 6, 11, 27, 29, 33, 48 residues to arginine) and HA-Ub K48 (mutation of lysine at 6, 11, 27, 29, 33, 63 residues to arginine) were synthesized by Mailgene Biosciences Co., Ltd.

The codon-optimized protein coding sequences (CDS) of SmCASP3/6/7/8 and SmGSDMEa/b were amplified by PCR. The CDS of SmGSDMEa-NT₂₆₂, SmGSDMEa-NT₂₀₂, SmGSDMEa-CT₂₆₂, SmGSDMEa-CT₂₀₂, SmGSDMEb-NT and SmGSDMEb-CT were subcloned from the above cloning sequence. Site-directed mutations of SmGSDMEa Asp202 to Ala (D202A), Asp259 to Ala (D259A), Asp262 to Ala (D262A), and mutation of SmGSDMEb Asp246 to Ala (D246A) were performed using the Hieff Mut Site-Directed Mutagenesis Kit (Yeasen, Shanghai, China). The recombinant plasmid was introduced into Trelief 5α (Tsingke Biological Technology, Beijing, China) by transformation. The mutations were verified by sequencing analysis. The primers used are listed in Table S1.