Disruption of one gadd7 allele in 2E1 cells leads to decreased
gadd7 RNA expression. A, schematic representation of
gadd7-retroviral fusion transcript with 5′ RACE primer location
and endogenous gadd7 transcript. B, directed PCR was
performed on cDNA from WT (lanes 1 and 2) and 2E1 (lanes
3 and 4) cells to detect gadd7 expression (lanes
1 and 3) and fusion transcript (lanes 2 and
4). Negative control reactions (lanes 5 and 6)
contained no cDNA. Forward (F) and reverse (R) primers for
gadd7 were used in PCR for lanes 1, 3, and 5.
Forward gadd7 primer and reverse primer for the proviral sequence
(ROSAβgeo) were used in PCR for lanes 2, 4, and 6. C,
basal gadd7 RNA expression in WT and 2E1 mutant cells was determined
by qPCR and normalized to β-actin RNA expression. *, p
< 0.05 for 2E1 versus WT. Data are expressed as mean ± S.E.
for three independent experiments. D, WT and 2E1 mutant cells were
treated with 500 μm palmitate for the indicated times and
gadd7 expression was determined by qPCR and normalized to
β-actin RNA expression. *, p < 0.05 for 2E1
versus WT; #, p < 0.01 for treated versus
untreated WT cells and for treated 2E1 versus treated WT. Data are
expressed as mean ± S.E. for three independent experiments.