A CHOP (C/EBP homologous protein) promoter-luciferase reporter in the pGL3 vector (CHOP-Luc) was provided by Dr Pierre Fafournoux (Unite de Nutrition et Metabolisme Proteique, INRA de Theix, Saint Genes Champanelle, France) (Bruhat et al, 2000).
The reporter vector pGL4.23-CHOP AARE and its mutant, non-responsive version pGL4.23-mutCHOP AARE, were generated by site-directed mutagenesis from pGL4.23[luc2/minP].
U2OS were transiently transfected with empty or dominant-negative CHOP (DN-CHOP) expression vector with lipofectamine 2000 (Invitrogen) and selected next day in G418 (400 µg/ml) for three days.
The expression vector for WT Chop was from Dr. Andreas Strasser, WEHI, Australia.
The vector did not induce CHOP expression in every hepatoma cell line (data not shown).
Cells were transfected with 2 μg of pGL4.17 promoter FGF21 (−1497/+5), 2 μg of ATF4, or CHOP expression vector using Fugene HD (Promega).
The expression vector containing the coding sequence of ATF4 or CHOP was preserved in our laboratory.
To ascertain the role of CHOP in cell death induction, we transiently transfected empty vector (ctr) or dominant-negative form of CHOP (DN-CHOP) in U2OS cells.
Interestingly, BiP, GADD34, CHOP, and ERp57 levels were also increased in HPNE vector cells treated with Reolysin, albeit to a much lesser degree than in KRas-expressing cells.
CHOP cDNA was obtained by PCR and cloned into pcDNA3.1 vector (Invitrogen, Freiburg, Germany) encoding an N-terminal Flag-tag.
The CHOP promoter-reporter construct was cotransfected into cells with either the 3a expression plasmid (or the vector control plasmid).
