Pspax2 and pmd2 g packaging plasmids

Lentivirus production was carried out as previously described (44 (link)). In brief, HEK293T cells were transfected with TransIT-LT1 (Mirus) in a 2:1:1 ratio of the lentiviral vector and psPAX2 and pMD2.G packaging plasmids (Addgene), according to the manufacturer's instructions. Viral supernatant was collected 48 and 72 hours after transfection. The supernatant was pooled, filtered, and stored at –80°C. Neuroblastoma and RPE cells were transduced with virus particles in the presence of 8 μg/mL hexadimethrine bromide (Merck). Cells were transduced for 1 day in antibiotic-free medium and then grown in the full medium for 1 day. Neuroblastoma cells were then selected for 2 days with puromycin hydrochloride (2 μg/mL) or geneticin disulfate (G418, Roth; 2 mg/mL).

For lentivirus production, HEK293T cells were transiently transfected with psPAX2 and pMD2.G packaging plasmids (Addgene) and corresponding expression vectors. Media were changed 24 h post transfection and replaced with the target cell line-specific media. After 48 h the supernatant (containing virus) was collected, filtered through a 0.45 μm filter supplemented with 8 μg/mL protamine sulfate (Sigma-Aldrich) and added to subconfluent target cells. Cells were then spinfected at 2000 rpm for 45 min at room temperature. After 24 h the media were exchanged with fresh media and 24 h later the media were complemented with selection antibiotic for 5–7 days to select infected target cells. Knockout and overexpression efficiency was evaluated by immunoblot analysis.

HEK293T cells in a 10-cm dish were transfected with PMD2G and PSPAX2 packaging plasmids (Addgene, Watertown, MA, USA), together with lentiviral-expressing vectors encoding target genes INPP4B (GeneCopoeia, Guangzhou, China). Supernatant carrying the viral particles was harvested after transfection. For viral infection, myeloma cells were seeded at (1−2) × 10⁶ cells per well in six-well plates and then added supernatant carrying the viral particles. Twelve hours after infection, the medium was changed, and cells were cultured for another 48 h until further management.

For lentivirus production 293T cells were plated in 10cm tissue culture dish and transfected with Lck^WT-mCherry, Lck^Y505F-mCherry or Lck^K273R-mCherry lentiviral plasmid and pMD2.G and psPAX2 packaging plasmids (Addgene) in the presence of polyethyleneimine. Culture supernatant containing lentiviral particles was harvested at 48 and 72 hours post-transfection and used to transduce the RLM mouse CD4 SP thymocyte cell line. Zap70 expression in transduced cells (mCherry+) was analysed using flow cytometry.

The HepG1, Hep3B, and H22 HCC tumor cell lines permanently transduced with SPINK1-OE or SPINK1-silencing (shRNA) lentiviral constructs were generally constructed as previously described (16 (link)). Briefly, the pLenti-CMV-hSPINK1-2A-GFP vector or pLenti-SPINK1 shRNA was co-transfected with either ABM’s Second Generation (LV003) Packaging Mix or the mixture of pMD2.G and psPAX2 packaging plasmids (Addgene Inc., Cambridge, MA, USA) into 293T cells, while the transfected empty or scrambled vector was used as a control. The packaged lentiviral particles were harvested 24–48 h after transfection. HepG2, Hep3B, and H22 cells were infected by filtered lentiviral particles, and the transduced cells were screened under pressure of 2 µg/ml puromycin for about 3 weeks before the drug-resistant cells were collected. Subsequently, the protein expression of SPINK1 was examined in passaged HepG1, Hep3B, and H22 cells using Western blotting.

BMS-754807 was purchased from MedChemExpress (Monmouth Junction, NJ, USA), CP from Accord (Durham, NC, USA), and recombinant human IGF-1 from PeproTech (Cranbury, NJ, USA). Protease inhibitor cocktail, sodium pyrophosphate, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent were purchased from ThermoFisher (Waltham, MA, USA). Hydroxychloroquine sulfate was purchased from Tokyo Chemical Industry (Portland, MA, USA). Sodium orthovanadate, β-glycerophosphate, isopropyl β-D-1-thiogalactopyranoside (IPTG), rapamycin, bafilomycin A1, and hygromycin were purchased from Sigma Aldrich (St. Louis, MO, USA). PMD2.G and psPAX2 packaging plasmids were purchased from Addgene (Watertown, MA, USA). ATG5 targeting (MSH091034-LVRU6H) and scramble control (CSHCTR001-LVRU6H) shRNA vectors were purchased from Vectorbuilder (Chicago, IL, USA).