Pcmv6 entry mammalian expression vector

The plasmid pCMV6-Entry/nestin was obtained by subcloning the full-length human nestin cDNA into the pCMV6-Entry mammalian expression vector (OriGene, Rockville, MD). The Nestin gene was under the control of the CMV promoter, allowing constitutive expression of nestin. SKLC17 cells (2 × 10⁵ cells/plate) were seeded in a 6-well plate and cultured for 24 h before transfection. Cells were transfected with pCMV6-Entry/nestin or control plasmids (20 μg) using the X-tremeGENE Transfection Reagent (Roche Applied Science, Indianapolis, IN) in serum-free Opti-MEM (Invitrogen, Carlsbad, CA) in accordance with the instructions from the manufacturer. After selection with G418 (500 μg/ml), proteins were extracted from each cell, and nestin expression was analyzed by Western blotting. After confirming increased expression of nestin, transfected cells were cloned in the presence of G418. Subsequently, two cell lines were selected from the transfected cells as nestin-overexpressing cell lines (SKLC17 NES-1 and NES-2). In the same way, cells transfected with the negative control plasmid were cloned, and one cell line that showed similar nestin expression to the parental cells was selected as the control cell line (SKLC17 ctrl). For the in vitro cell proliferation assay, 1 × 10⁴ cells were cultured in 6-cm dishes. Cells were trypsinized and counted after 48, 72, and 96 h.

Murine Aim2 or Asc CDS sequences were cloned into pCMV6‐Entry Mammalian Expression Vector (Origene). HEK 293T cells were transiently transfected with AIM2/ASC or ASC plasmids using Lipofectamine 3000 transfection reagent (#L30000015, Invitrogen) according to the manufacturer's instructions.