Fig. 3.

Decreased GATA6 expression is associated with inhibition of mammalian target of rapamycin (mTOR). A: 16 h after pretreatment with 75 nM torin 1 or 25 nM rapamycin (rapa), human aortic endothelial cells (HAECs) were stimulated with TNF-α for the indicated time followed by Western blot analysis for transcription factors (n = 3). The 0-, 10-, and 30-min TNF-α treatments were in the same blot. B: HAECs were pretreated with torin 1 or rapamycin and stimulated with TNF-α for 30 min. The nuclear extract was submitted for Western blot analysis of transcription factors (n = 3). C: HAECs were treated as described in Fig. 2A and ICAM1 mRNA was quantified (n = 5–6). D and E: HAECs were pretreated with torin 1 or rapamycin and stimulated with TNF-α for 4 h followed by Western blot analysis of the whole cell lysate (D) or nuclear extract (E) for interferon regulatory factor 1 (IRF-1) and GATA6 (n = 3). F: IRF-1 luciferase reporter activity was measured by the Dual-Luciferase Reporter system (n = 4). G and H: HAECs were grown on coverslips and immunolabeled for GATA6. Images were obtained by fluorescence microscopy (G) and nuclear intensity quantified (H) (n = 4). I: HAECs were transfected with siRNA and stimulated with TNF-α for 4 h followed by Western blot analysis of transcription factors (n = 5). Values are means ± SE. ctrl, control; MFI, mean fluorescence intensity; p-, phosphorylated; siCtrl, control siRNA; siMTOR, mTOR-targeted siRNA; siRAP, regulatory-associated protein of mTOR (RAPTOR)-targeted siRNA; siRIC, rapamycin-insensitive companion of mTOR (RICTOR)-targeted siRNA. *P ≤ 0.05; **P ≤ 0.001; ***P ≤ 0.0001 (one-way ANOVA followed by Dunnett’s test, C and H; two-tailed paired t-test, F).