FIGURE 6.
gadd7 is a regulator of palmitate-induced ROS generation. A, WT cells were treated with 500 μm palmitate in the presence or absence of vitamin E (Vit E) for 12 h. ROS generation was quantified by CM-H2DCFDA labeling and flow cytometry. *, p < 0.001 palmitate (Palm)-treated versus untreated; #, p < 0.01 Vit E/palmitate-treated versus Vit E-treated; ‡, p < 0.05 Vit E/palmitate-treated versus palmitate-treated cells. B, WT cells were treated as in A, and gadd7 expression was determined by qPCR normalized to β-actin RNA expression. *, p < 0.001 palmitate-treated versus untreated; #, p < 0.05 Vit E/palmitate-treated versus Vit E-treated; ‡, p < 0.005 for Vit E/palm-treated versus palmitate-treated cells. C, WT cells were treated with 500μm palmitate in the presence or absence of NAC for 12 h. ROS generation was quantified as in A.*, p < 0.001 palmitate-treated versus untreated; #, p < 0.001 NAC/palmitate-treated versus NAC-treated; ‡, p < 0.05 NAC/palmitate-treated versus palmitate-treated cells. D, WT cells were treated as in C, and gadd7 expression was determined by qPCR as in B.*, p < 0.001 palmitate-treated versus untreated; #, p < 0.001 NAC/palmitate-treated versus NAC-treated; ‡, p < 0.001 for NAC/palmitate-treated versus palmitate-treated cells. E,WTand 2E1 cells were treated with palmitate and analyzed for ROS generation as in A.*, p < 0.05 for treated versus untreated mutant cells; #, p < 0.001 for treated mutant versus treated WT cells. F, stable scrambled (SCR) and gadd7 knockdown (KD1 and KD2) cell lines were treated with palmitate and analyzed for ROS generation as in A.*, p < 0.001 for treated versus untreated knockdown cells; #, p < 0.005 for treated knockdown versus treated SCR cells. All data are expressed as mean ± S.E. for three independent experiments.