Blasticidin selection

Macrophage colony-stimulating factor (M-CSF)-dependent DGCR8^f/f bone marrow macrophages (BMMs) were prepared from the femurs and tibias of 4-to-6-week-old DGCR8^f/f mice and Cre-containing retroviruses were infected into the cells as described previously [Sugatani et al., 2011 (link)]. After two days blasticidin selection (2 μg/mL) (Invitrogen), the infected cells were expanded and harvested for following assays.

Human PRSS3 complementary DNA (cDNA) (sequence identification number NM_007343.3) was amplified by PCR and cloned into the plenti6-GFP vector (Invitrogen). PRSS3-expressing lentiviral or empty vectors were packaged using the ViraPower™ lentiviral expression system (Invitrogen, San Diego, CA, USA). The resulting lentivirus was used to infect PLC/PRF/5 or HepG2 cells and was subjected to blasticidin selection (2 μg/ml, Invitrogen) for 2 weeks to generate stable cell lines expressing PRSS3.

The THOR expression construct were generated by amplifying the full-length transcript from NCI-H1299 cells and subcloning into the pLenti6 expression vector (Invitrogen), LacZ constructs were used as controls. Following Sanger sequencing (University of Michigan Sequencing Core) confirmation of the inserts, lentiviruses were generated at the University of Michigan Vector Core. NCI-H1437 and SK-MEL-5 cells were infected with lentiviruses expressing THOR or LacZ and stable pools and clones were generated by blasticidin selection (Invitrogen). The THOR deletion constructs were also generated by amplifying by PCR using the full-length transcript as a template and were subcloned into the pLenti6 expression vector (Invitrogen).