Cell culture, vector construction, and gene knockout

Human cervical cancer cell line (HeLa) (ATCC^® CCL-2™, Cedarlane) and HEK293 stably expressing the 6xHis-SUMO3-Q87R/Q88N mutant (HEK293-SUMO3m)²⁷ were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum (FBS; Wisent), 1% l-glutamine (Thermo Fisher Scientific), and 1% penicillin/streptomycin (Invitrogen) in 5% CO₂ at 37 °C.

The mammalian expression vector for Myc-PIAS1 was constructed by inserting the full-length cDNAs into pcDNA3.0-Myc. The mammalian expression vector for PIAS1-GFP-WT was constructed by cloning full-length PIAS1 cDNA into pcDNA3.1-c-GFP10. The mammalian expression vectors pReceiver-M11 (Flag-NSMCE2, Flag-PFDN2, Flag-VIM, and Flag-empty control) were purchased from Genecopoeia, Inc. (Rockville, MD). The mammalian expression vector Emerald-VIM was purchased from addgene (#54300). The PIAS1-GFP-K137R, PIAS1-GFP-K238R, PIAS1-GFP-K315R and PIAS1-GFP-3XKR, Flag-VIM^mt, and Emerald-VIM^mt plasmids were generated by site-directed mutagenesis using the GeneArt Site-Directed Mutagenesis System, according to the instructions of the manufacturer (Invitrogen^TM). The PIAS CRISPR/Cas9-based gene KO vectors pCRISPR were also purchased from Genecopoeia, Inc. (Rockville, MD). Primer sequences used for vector construction, site-directed mutagenesis, and sgRNA sequences used for CRISPR/Cas9 gene KO are listed below: PIAS1_F: 5′-GGGTACCATGGCGGACAGTGCGGAAC-3′, PIAS1_R: 5′-GGAATTCTCAGTCCAATGAAATAATGTCTGG-3′, PIAS1_K137R_F: 5′-GTCCATCCGGATATAAGACTTCAAAAATTACCA-3′, PIAS1_K137R_R: 5′-TGGTAATTTTTGAAGTCTTATATCCGGATGGAC-3′, PIAS1_K238R_F: 5′-TACCTTCCACCTACAAGAAATGGCGTGGAACCA-3′, PIAS1_K238R_R: 5′-TGGTTCCACGCCATTTCTTGTAGGTGGAAGGTA-3′, PIAS1_K315R_F: 5′-GCTTTAATTAAAGAGAGGTTGACTGCGGATCCA-3′, PIAS1_K315R_R: 5′-CGGATCCGCAGTCAACCTCTCTTTAATTAAAGC-3′, VIM_K439R_F: 5′-GTTGATACCCACTCAAGAAGGACACTTCTGATT-3′, VIM_K439R_R: 5′-AATCAGAAGTGTCCTTCTTGAGTGGGTATCAAC-3′, VIM_K445R_F: 5′-AGGACACTTCTGATTAGGACGGTTGAAACTAGA-3′, VIM_K445R_R: 5′-TCTAGTTTCAACCGTCCTAATCAGAAGTGTCCT-3′, PIAS1-sgRNA: 5′-TTCTGAACTCCAAGTACTGT-3′, PIAS2-sgRNA: 5′-CAAGTATTACTAGGCTTTGC-3′, PIAS3-sgRNA: 5′-GCCCTTCTATGAAGTCTATG-3′, PIAS4-sgRNA: 5′-GGCTTCGCGCCGTAGTCTTAG-3′, and scrambled sgRNA: 5′-GGCTTCGCGCCGTAGTCTTA-3′.

For transient transfection, cells were transfected with 1 μg plasmid per million cells using JetPrime Reagent (Polyplus transfection) according to the manufacturer’s protocol. Cells were collected 36 h or 48 h after transfection for further experiments and protein overexpression was confirmed by western blotting.

For PIAS gene KO, cells were transfected with 1 μg plasmid per million cells using JetPrime Reagent (Polyplus transfection) according to the manufacturer’s protocol. Cells were sorted by fluorescence-activated cell sorting based on Red fluorescent proteins-mCherry signals 48 h after transfection. mCherry-positive cells were cultured and expended for another week (Supplementary Fig. ¹). PIAS KO efficiency was confirmed by western blotting.