Pspax2

Lentiviral expression vectors were co-transfected into HEK293FT cells with the lentiviral packaging plasmids psPAX2 and pMD2.G (#12260 and #12259; Addgene). In brief, 3 × 10⁶ HEK293FT cells were seeded into a poly-L-lysine-coated 10-cm culture dish the day before transfection. For each 10-cm dish, the following DNA was diluted in 0.6 ml of OptiMEM (Thermo Fisher Scientific): 4.5 μg of lentiviral vector, 6 μg of psPAX2 and 1.5 μg of pMD2.G. Separately, 35 μL of 1 μg/μL 25 kDa polyethylenimine (PEI; Sigma) was diluted into 600 µL of OptiMEM, briefly vortexed and incubated at room temperature for 5 min. After incubation, the DNA and PEI mixtures were combined, briefly vortexed and incubated at room temperature for 20 min. During this incubation, the culture medium was replaced with 8 ml of pre-warmed DMEM + 1% FBS. The transfection mixture was then added dropwise to the 10-cm dish. Viral particles were collected 48 h after the medium change and filtered through a 0.45-μm PVDF syringe filter. The filtered supernatant was used directly to infect cells. Aliquots were snap-frozen and stored at −80 °C. For transduction, lentiviral particles were diluted in complete growth medium supplemented with 10 μg/ml polybrene (Sigma) and added to cells.

A lentiviral packing system including plasmids pMD2.G and psPAX2 (Sigma) was utilized to knock down gene expression via delivering shRNA into the target cells. All lentiviral shRNA plasmids were derived from a pLKO.1‐puromycin backbone. shRNA plasmids of JunD, Pbk, and scramble shRNA were obtained from the University of Pennsylvania Perelman School of Medicine High‐Throughput Screening Core. Specific shRNA sequences can be found in Appendix Table S3.
LentiCRISPRv2 was obtained as a gift from Dr. Anil Rustgi for use to knock out genes via a CRISPR technique. The lentiCRISPRv2 vector with Men1 targeted sgRNA was sequenced to confirm that the correct insert was in place. Specific sgRNA sequences are in Appendix Table S3. To produce lentivirus, HEK293T cells were transfected with pMD2.G, psPAX2, and the sgRNA expression plasmid of interest using the calcium chloride precipitation method as previously reported (Katona et al,2019).
A pMX‐puro‐Pbk construct, as previously published, and a mutant plasmids with K64K65 to AA based on pMX‐puro‐Pbk construct, were transfected into INS‐1 cell line to induce ectopic expression of Pbk.
Cells were transduced by the virus of choice in the presence of 4 μg/ml polybrene (hexadimethrine bromide) for lentivirus infection. 24 h after completion of transduction, cells were then selected with puromycin for 72 h.

PTPRD shRNA (bacterial glycerol stocks of five different sequences) and the non-targeting scrambled shRNA, pLKO.1-puro, were purchased from Sigma Aldrich. Each DNA sample (2 μg) was co-treated with psPAX2 (Sigma Aldrich, 1.5 μg), pMD2.G (Sigma Aldrich, 0.5 μg), and E-fection (2 μg/μg DNA) for lentiviral production in 293TN cells. Viruses were harvested at 24 and 48 h. Lentiviral constructs and 8 μg/mL polybrene (Chemicon, Billerica, MA, USA) were used to transfect MKN74 cells, which were then selected with puromycin (1 μg/mL), to generate stable cell lines. The efficiency of PTPRD mRNA knockdown by the five different shRNA sequences was tested by qRT-PCR and shPTPRD #3 was chosen for subsequent experiments.