Hcas9

PODXL1-knockout PDAC cell lines (for MIaPaCa-2, AsPC-1, and Panc-1) were generated using CRISPR/Cas9 system. Both plasmids, hCas9 (#41815 Addgene, Watertown, MA), and gRNA (guide RNA) Cloning Vector (#41824), were obtained from Addgene. The gRNA vector including PODXL1 target sequence (CGACACGATGCGCTGCGCGCtgg) located in part of the Exon 1 was prepared following the manufacturer’s instruction with “tgg” sequence as a Proto-spacer Adjacent Motif (PAM). The hCas9 and PODXL1 gRNA vector were cotransfected into cells using ViaFect™ Transfection Reagent (#E4981, Promega, Madison, WI). Twenty-four hours posttransfection, the cells were cultured with RPMI medium containing 500 μg/ml of Geneticin (#10131-35, Gibco, Thermo Fisher Scientific, Waltham, MA) for isolating the Geneticin-resistant clones. PODXL1-expression deficient clones from each PDAC line were confirmed by lack of PODXL1 protein, using immunoblot analysis with anti-PODXL1 antibody. Genetic mutation of PODXL1 in the knockout clone was also examined by genomic DNA sequencing of PCR-amplified product, using the specific primers for PODXL1-target sequence and the adjacent genomic DNA. The primers used for the sequencing were the following: Pod1.Ex1.check.Fw2: 5′-CAGCGGCAGGGAGGAAGAGC and Pod1.Ex1.check.Rv2 5′-GCGGTGCGGTCTCCCTTTTCTT.

HeLa cells and mouse embryonic fibroblasts (MEFs) were maintained in DMEM supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin mix in a humidified 37°C incubator with 5% CO₂. HeLa cells were seeded at 100,000 cells in 2 ml medium per well in a six-well dish; transfected with 333 ng each of hCas9 (plasmid 41815; Addgene; Mali et al., 2013 (link)), gRNA_AAVS1-T2 (plasmid 41818; Addgene; Mali et al., 2013 (link)), and AAVS1_Puro_PGK_mCherry-progranulin (Nguyen et al., 2018 (link)) plasmids added to the 100 µl of Opti-MEM and 3 µl Fugene 6 transfection mix; and preincubated for 20 min. 48 h after transfection, the transfected cells were selected with puromycin (2 µg/ml) for 2 wk to isolate the surviving cells that stably expressed mCherry-progranulin. An immortalized line of CLN6 mutant MEFs (as well as a WT littermate controls) was generated by serial passaging of primary MEFs from the nclf line of spontaneous mutant mice obtained from The Jackson Laboratory (Todaro and Green, 1963 (link); Gao et al., 2002 (link)).

HeLa/5×Flag-RNase L cells were obtained as follows. HeLa cells were transfected with hCas9 (Addgene plasmid # 41815), pgRNA-RNase L and p5×Flag-RNase L-Donor. Two days after transfection, cells were selected with 0.5 μg/ml puromycin and maintained as a polyclonal pool.