Plenti6 tr vector

To promote the HMGB1 level in PC12 (Syn^wt) or PC12 (Syn^mt) cells, the coding sequence of HMGB1 or the coding sequence of Red Fluorescence Protein (RFP) was cloned into the pcDNA3.1(+) vector. And 5 μg/mL (to guarantee more than 90 % cells to be transfected) HMGB1-pcDNA3.1(+) or RFP-pcDNA3.1(+) plasmid was transfected into 10⁵ per well 85 % confluent PC12 (Syn^wt) or PC12 (Syn^mt) cells. To upregulate the Beclin1 level in PC12 cells, Beclin1 or the coding sequence of Chloramphenicol acetyl transferase(CAT) was amplified and was cloned into the pLenti 6/TR vector (Invitrogen, Carlsbad, CA, USA). Recombinant pLenti-Beclin1 (LV-Beclin1) or pLenti-CAT virus of (LV-Con) was produced by cotransfecting 293 T cells with pLenti-Beclin1 or pLenti-CAT and ViraSafe™ Lentiviral Packaging System (Cell Biolabs, San Diego, CA, USA). PC12 (Syn^wt) or PC12 (Syn^mt) cells were infected with LV-Beclin1 or LV-Con virus with 1 Multiplicity of infection (MOI) for 12 or 24 hours.

The pLenti6/TR vector (Invitrogen, Cat. No. V48020) and pLenti3.3/TR vector (Invitrogen, Cat. No. A11114) were used to generate stable cell lines that constitutively express high levels of the Tet repressor. These expression plasmids contain elements that allow packing of the construct into virions and the blasticidin or geneticin resistance marker for selection of stably transduced cell lines. Briefly, 293FT packaging cells (Invitrogen, Cat. No. R700-07) were transfected with the pLenti6/TR or pLenti3/TR and the ViraPower^TM Packaging Mix (Invitrogen, Cat. No. K4975-00) to produce lentiviral particles. The lentivirus were used to transduce AGS cells or MGC-803 and the blasticidin (8 µg/ml, Invitrogen, Cat. No. R210-01) or geneticin (200 µg/ml, Invitrogen, Cat. No. 10131-027) was used to select for a stable TR-expressing AGS cells named TR-AGS stable cell line or TR-expressing MGC-803 cells named TR-MGC-803 stable cell line. These TR expressing cell lines were used as hosts for expression constructs that facilitate Tet-regulated expression of CDH17 shRNA (short hairpin RNA) or CDH17 gene, respectively.