Polyjet transfection reagent

Plasmid transfection for lentivirus production was performed using a Poly-jet transfection reagent according to the manufacturer’s protocol (SignaGen Laboratories, Toronto, ON, Canada). In brief, 1 × ${10}^6$ HEK293T cells (passage number lower than 10) were seeded in each 35 mm cell culture plate pre-coated with 250 µL of 0.1 mg/mL poly-L-lysine. The next day, the growth media in each plate were replaced with prewarmed fresh media 30 min prior to transfection. Then, 0.5 µg of each of the pTRIPZ-PTPs, or Lenti-iCas9-neo or gPTPN12 in LentiGuide plasmid, was mixed with 0.38 µg of psPAX viral packaging plasmid, 0.13 µg of pMD2G viral envelop plasmid, and 3 µL of PolyJet transfection reagent (SignaGen, # SL100688) in serum-free DMEM medium and incubated at room temperature for 15 min. The mixture was added dropwise into the medium containing cells. The next day, the medium was replaced with 1 mL of DMEM/10%FBS containing 10 mM of Sodium Butyrate (Santa Cruz Biotechnology, Santa Cruz, CA, USA # sc-202341A). Sodium Butyrate induces gene expression in cells. About 24 h after the treatment of the cells with Sodium Butyrate, the lentivirus-containing media were collected and aliquoted into 250 µL per 1.5 mL Eppendorf tube. After flash-freezing in liquid nitrogen, all virus aliquots were kept at −80 °C.

The PUMA promoter (-150 bp to +50 bp relative to transcription start site containing the AP-1 binding site) (Cazanave et al., 2009 (link)) was synthesized and cloned into pGL-3 basic vector (Promega). The 4X AP-1 luciferase reporter plasmid was as previously described (Wang et al., 2008 (link)). Cells (3 × 10⁴ cells per well in a 24-well plate) were transfected with 0.2 μg of PUMA promoter luciferase reporter or 0.2 μg of 4X AP-1 luciferase reporter along with 10 ng of Renilla luciferase reporter (pRL-SV40) using PolyJet transfection reagent (SignaGen Laboratories). Cells were lysed in 1 × passive buffer (Promega) and the bioluminescent signal was determined with 20/20 luminometer using Dual-Luciferase Reporter Assay kit (Promega).

HepG2 cells were seeded in 96-well culture plate (3 × 10⁴cells/well) and transfected with 100 ng of p-hTRIB1-Luc plasmid or pGL3-basic vector, and co-transfected with 10 ng of pCMV-β-Gal as an internal control by using Polyjet transfection reagent (SignaGen Laboratories, Gaithersburg, MD). One day post-transfection, cells were starved overnight in 0.5% FBS containing MEM and subsequently treated with BBR at a dose of 40 μM for 8 h or 24 h in the absence or presence of MEK1 inhibitor U0126. At the end of treatment time course, cells were lysed with 100 μl reporter lysis buffer with 50 μl cell lysate for measuring β-galactosidase activity by using β-Galactosidase Enzyme Assay System (Promega) and the remaining 50 μl of lysate for firefly luciferase activity assay (Luciferase Assay System (Promega). Absolute luciferase activity was divided by β-galactosidase activity to correct for transfection efficiency. Triplicate wells were assayed for each transfection condition.