Fig. 5.

HF Inhibition of cytokine responses is sensitive to depletion of GCN1, but not GCN2. (A) Characterization of protein expression and GCN2 phosphorylation in GCN1- and GCN2-depleted K4 cells. K4 were transfected with a lentiviral vector carrying two independently targeted shRNAs against GCN2 (#2 or #3) or GCN1 (#698 or #699), and protein expression and GCN2 phosphorylation examined by Western blot 15 min after HF treatment. Scrambled shRNA (SC) infected cells and uninfected cells were used as negative controls. (B) Evaluation of knockdown effects on induction of the AAR marker Trib3. GCN2 or GCN1 knockdown cells were treated with HF (100 nM) and/or TNF-α (10 ng/mL). For transcript levels of TRIB3, cells treated with HF for 6 h. (C) Evaluation of knockdown effects on HF inhibition of TNF-α–induced genes. For transcript levels of MMP13 (Upper) or CXCL10 (Lower), cells were pretreated with HF and treated with TNF-α for 6 h. Transcript levels of target genes were quantified by qPCR, Results are representative of three independent experiments (means ± SD, n = 3). **P < 0.01, ***P < 0.001; ns, not significant.