Figure 6. Pharmacological inhibition of TBK1 impairs AKT signaling.
(A) Structure of Compound II.
(B) IC50 values for in vitro inhibition of the indicated purified recombinant kinases by Compound II.
(C) Primary macrophages from mouse bone marrow were treated with LPS and increasing concentrations concentration of Compound II. LPS induced accumulation of interferonβ (IFNβ) and interferonβ target gene (IP10 and Mx1) mRNAs were measured by quantitative PCR and shown as percent of inhibition. Error bars represent SDM from triplicate experiments.
(D) HeLa cells incubated in the indicated concentrations of Compound II were stimulated with 10 ng/ml TNFα for 10 minutes (p65 assays), or transfected with poly I:C for 2 hours (IRF3 assays), followed by immunofluorescence-based detection of IRF3 and p65 nuclear accumulation. Nuclear accumulation is plotted as percent of control (POC). Error bars represent SDM from triplicate analysis.
(E) Wild-type MEFs were incubated cells in DMEM with 10% serum but without glucose for 2 hours. Cells were then pretreated with 2 µM Compound II for 30 minutes as indicated followed by addition of 25 mM glucose as indicated. Whole cell extracts were prepared post glucose stimulation and immunoblotted as shown. (Molecular size: as described)
(F) Asynchronous proliferating cultures of HCC44 cells were exposed to the indicated concentrations of Compound II for 30 minutes. Whole cell extracts were immunoblotted for detection of the indicated proteins and phospho-proteins. (Molecular size: as described)
(G) Sin1−/− and Rictor−/− MEFs were pretreated with DMSO or Compound II for 30 minutes as indicated, followed by exposure to LPS (1 µg/ml) or Sendai virus (SeV, 100 HA/ml). Whole cell extracts prepared at the indicated time-points were immunoblotted for detection of AKT activation. (Molecular size: as described)
(H) Whole cell extracts from HCC44, A549 and Mia-Paca2 cells exposed to the indicated concentrations of Compound II or LY294002 for 24 hours were immunoblotted to detect consequences on AKT pathway activation. (Molecular size: cleaved-PARP: 89KDa; others as described)