Plasmid kit

Restriction enzyme digests, DNA ligations, and other recombinant DNA procedures were performed using standard protocols. Mutagenesis was performed using the QuikChange site-directed mutagenesis method (Stratagene) with the KOD polymerase (Novagen). DNA constructs used for transfection were purified from E. coli DH5α using Qiagen Plasmid kits according to the manufacturer’s protocol. All DNA constructs were sequence verified. Lysis buffer used for purifying proteins from insect cells was composed of 50 mM Tris-HCl (pH 7.6), 300 mM NaCl, 20 mM imidazole, 5% (v/v) glycerol, 0.075% (v/v) 2-mercaptoethanol, 1 mM benzamidine, 1 mM PMSF. Lysis buffer was supplemented with SigmaFast protease inhibitors (EDTA-free) at 1 tablet/100 ml (Sigma). Wash buffer A was the same as lysis buffer without PMSF and protease inhibitors. High salt wash buffer B was the same as buffer A with 500 mM NaCl. All protein concentrations were calculated using the Beer-Lambert law by measuring the absorbance at 280 nm. Theoretical extinction coefficients were calculated using the ProtParam tool at the ExPASy proteomics server (http://www.expasy.org).
See Supplementary Information for a detailed description of the methods and materials used in this study.

A human prelamin A cDNA in pCMV-XL5 vector (#SC101048) was purchased from Origene (Rockville, MD). A human progerin cDNA was created by deleting 150 nucleotides (1818–1968) from the prelamin A cDNA with the QuikChange Lightening kit (Agilent; Santa Clara, CA) and mutagenic primers (forward primer, 5′-GCTCAGGAGCCCAGAGCCCCCAGAACTG-3′; reverse primer, 5′-CAGTTCTGGGGGCTCTGGGCTCCTGAGC-3′). The doxycycline-inducible vector pTRIPZ-hDDX5/17 (Addgene; Cambridge, MA) was digested with restriction enzymes AgeI and EcoRI to delete the red fluorescence protein and shRNA sequences and then gel-purified. The human prelamin A and progerin cDNAs were amplified with the Titanium Taq PCR kit (Clontech; Mountain View, CA) and sequence-specific primers (forward primer, 5′-GTCAGATCGCACCGGATGGAGACCCCGTCCCAG-3′; and reverse primer, 5′-GTAGCCCCTTGAATTTTACATGATGCTGCAGTTCTGGGG-3′). The fragments were purified with UltraClean15 (Mo-bio) and subcloned into the prepared pTRIPZ vector with In-Fusion Cloning (Clontech). The products were amplified in XL10-Gold Ultracompetent cells (Agilent) and plasmids with the correct sequence were isolated with plasmid kits (Qiagen; Germantown, MD). Packaging of lentivirus and transduction of cells were performed by UCLA’s Vector Core. Transduced cells were selected for two weeks with 1.5 μg/ml puromycin.

The EGFP-KASH2 sequence was amplified from pEGFP-C1-KASH2 (50 (link)) with the TaKaRa LA PCR kit (Clontech) and sequence-specific primers (forward primer, 5′-ACACCGACTCTAGAGATGGTGAGCAAGGGCGAGGA-3′; and reverse primer, 5′-GCGGGCCCTCTAGACCTAGGTGGGAGGTGGCCCGT-3′). The EGFP-KASHext sequence was amplified from pEGFP-C1-KASHext (50 (link)) with the TaKaRa LA PCR kit (Clontech) and sequence-specific primers (forward primer, 5′-ACACCGACTCTAGAGATGGTGAGCAAGGGCGAGGA-3′; and reverse primer, 5′-GCGGGCCCTCTAGACTTATCTAGATCCGGTGGATC-3′). The gel-purified fragments were subcloned into the pLenti6/v5-DEST plasmid (ThermoFisher) (linearized with BamHI and XhoI), with In-Fusion Cloning (Clontech). The products were amplified in XL10-Gold Ultracompetent cells (Agilent) and plasmids with the correct sequences were isolated with plasmid kits (Qiagen; Germantown, MD). Packaging of lentivirus and transduction of pTRIPZ-prelamin A or pTRIPZ-progerin cells were performed by UCLA’s Vector Core. Transduced cells were selected for two weeks with 2 μg/ml blasticidin.