Miniprep kit

Plasmids were amplified by PCR (14 cycles) with mutant primers and cut with DpnI enzyme (NEB, USA) followed by enzyme deactivation at 85 °C. After enzyme deactivation, the plasmids were transformed into DH5alpha competent cells (Enzynomics, Korea). Cloned plasmids were obtained using a Miniprep kit (Intron Biotechnology, Korea). Mutants were confirmed by DNA sequencing (Cosmo Genetech, Korea). Primer lists used in cloning are provided in Table S3.

Traditional cloning using restriction enzymes and T4 DNA ligase was used to clone the FLAG-tagged ZNF212 expression vector and luciferase vector containing the Pld3 promoter. The human ZNF212 open reading frame (ORF) construct was purchased from Origene (USA), and the promoter of human Pld3 originated from the genomic DNA of the human SH-SY5Y cell line. Human ZNF212 ORF and PLD3 promoters were amplified by PCR (35 cycles) using a KAPA HIFI PCR kit (KAPAbiosystem, USA). Amplified PCR products were cloned into the cut and ligation protocols. Amplified DNA fragments and empty vectors (pCMV-tag2A and pGL3-basic, respectively) were cut using restriction enzymes (NEB, USA) at 37 °C for 3 h and separated on a 0.8% agarose gel (Bioshop, USA)/0.5% tris–acetate EDTA buffer, and then purified with a Gel/PCR clean-up kit (Intron Biotechnology, Korea) according to the manufacturer's protocol. Purified DNA was ligated with T4 DNA ligase (NEB, USA) at 16 °C overnight and transformed into DH5alpha competent cells (Enzynomics, Korea). Cloned plasmids were obtained using the Miniprep kit (Intron Biotechnology, Korea) and sequenced (Cosmogenetech, Korea) for validation.

IL-32θA94V coding sequence was synthesized by Bioneer (Daejeon, Korea) using HT-oligo™ synthesis. The synthesized DNA sequence was cloned into a pTH24 based TEVSH vector (Addgene, Watertown, MA, USA) using NdeI and AgeI restriction sites and rapid DNA ligation kit (Thermo Fisher Scientific, Waltham, MA, USA). TEVSH was a gift from Helena Berglund (Addgene plasmid # 125194; http://n2t.net/addgene:125194; RRID : Addgene 125194) and confirmed by DNA sequencing (Bionics, Seoul, Korea). The recombinant TEVSH vectors were transformed into DH5α by heat shock and purified using a mini prep kit (Intron Biotechnology, Sungnam, Korea). The IL-32θA94V expression vectors were transformed into Rosetta strain of Escherichia coli by heat shock transformation. Successfully transformed single colony was picked up and cultured at 37°C for 4 h in a 200-rpm incubator in Luria–Bertani (LB) media with ampicillin (100 μg/mL). The cultured mixture was transferred to 2.4 L of fresh LB media with ampicillin (100 μg/mL) and grown in a 200-rpm incubator at 37°C until it reached an OD₆₀₀ of 0.6. IL-32θA94V was induced by adding 0.5 mM IPTG. Cells were grown in a 200-rpm incubator at 16°C for 16 h.