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. 2017 Nov 17;37(1):19–38. doi: 10.15252/embj.201696164

Figure 5. TLR3 and TLR4 promote mTORC1 signaling in a TBK1‐dependent manner in RAW264.7 macrophages and primary bone marrow‐derived macrophages (BMDMs).

Figure 5

  1. Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 and TLR4 in cultured macrophages. RAW264.7 cells cultured in full serum were pre‐treated with amlexanox [50 μM or 100 μM] (2 h), rapamycin [20 ng/ml] (30 min), or Ku‐0063794 [1 μM] (30 min) and stimulated −/+ poly(I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min). Whole‐cell lysate (WCL) was immunoblotted as indicated.
  2. Knockdown of TBK1 with shRNA reduces TLR3‐ and TLR4‐stimulated mTORC1 signaling. RAW264.7 macrophages were co‐infected with shRNA‐containing lentiviruses targeting TBK1 and IKKε shRNA or infected with scrambled control (Scr), selected in puromycin, and treated as in (A).
  3. Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 and TLR4 in primary macrophages. BMDMs were pre‐treated with amlexanox [100 μM] (2 h) or Ku‐0063794 [1 μM] (30 min) and stimulated −/+ poly(I:C) or LPS as in (A). The arrow indicates S6K1 phosphorylated on T389.
  4. Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 in HEK293‐TLR3 cells. Cells were serum‐starved (20 h), pre‐treated with amlexanox or Ku‐0063794 as in (C) above, and stimulated −/+ poly(I:C) [50 μg/ml] (60 min).
  5. TLR4/LPS‐stimulated mTORC1 signaling requires TBK1. TBK1+/+ and TBK1−/− MEFs were serum‐starved (20 h), pre‐treated with amlexanox [50 μM] (2 h) or Torin1 [100 nM] (30 min), and stimulated −/+ LPS as in (A).