Plenti puro

The wild-type and phosphorylation-site mutant SKA3 lentiviral expression plasmids were generated by inserting wild type or mutant SKA3 CDS fusion with a Flag tag sequence into the BamHI and XhoI sites of the lentiviral vector pLenti-puro (a gift from Ie-Ming Shih, Addgene plasmid # 39481). For SKA3-knockdown stable cell generation, three sgRNAs targeting SKA3 exon 1 were synthesized and inserted into the pSpCas9(BB)-2A-Puro vector (Addgene plasmid # 62988). shRNA constructs targeting the top 50 upregulated genes used for high-content screening and the negative-control construct were purchased from Sigma-Aldrich (Munich, Germany). Wild-type and phosphorylation-site mutant SKA3 transient expression plasmids were constructed by inserting the corresponding expression frame into p3×FLAG-CMV-10 vector (Sigma-Aldrich). PLK1, PTEN, and Ubiquitin (Ub) expression plasmids were generated by inserting coding sequence into pCMV-HA vector (Clontech). Luciferase reporter plasmid pGL4.10-SKA3 was generated by inserting the promoter sequence (+100 to −1000 relative to transcription start site) into pGL4.10 vector. Transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.

Human SAR1 was subcloned into pLenti-puro (Addgene, Cambridge, MA, USA; plasmid 39481). The plasmid containing SAR1 was transfected with Lipofectamine 2000 (Invitrogen) following the manufacturer's recommendations into HEK293T cells at 50% confluence on the day of transfection along with the lentiviral packaging plasmids pVSVg (3.5 μg) and psPAX2 (6.5 μg, Addgene). Transfection was performed using one 10-cm dish. After 24-h transfection, the medium was changed, and after an additional 24 h, the medium was removed and filtered through a 0.45-μm low-protein binding membrane (VWR International, Radnor, PA, USA). McArdle-RH7777 or IMR-90 cells were then transduced with the virus with 8 μg/ml Polybrene (Sigma-Aldrich). After 24 h, the medium was replaced with fresh medium, and after an additional 24 h, 2 μg/ml puromycin (Sigma-Aldrich) was added to select transduced cells.