Fig. 6

FFAR2-knockout lung cancer cells exhibit enhancement of the cAMP-AMPK-TAK1 signaling axis for the activation of NF-κB in response to TLR2 or TLR3 stimulation. A and B. FFAR2KO A549 (A) or FFAR2KO H1299 (B) cells were generated using the CRISPR/Cas9 method. The expression of FFAR2 was evaluated by Western blotting. C-E. Ctrl A549 and FFAR2KO A549 cells were treated with vehicle, HKLM, and poly(I:C). The activation of AMPK and TAK1 was evaluated by pho-AMPK and pho-TAK1 antibodies (C). The band intensity of pho-AMPK (D) and pho-TAK1 (E) was measured using ImageJ, and normalized with the band intensity of vehicle treatment. The results are presented as means ± standard deviation (SD, n = 3 independent experiments). \(^{\blacklozenge }\)P < 0.05 and \(^{\blacklozenge \blacklozenge}\)P < 0.01. (F-H). Ctrl H1299 and FFAR2KO H1299 cells were treated with vehicle, HKLM, and poly(I:C). The activation of AMPK and TAK1 was evaluated by pho-AMPK and pho-TAK1 antibodies (F). The bend intensity of pho-AMPK (G) and pho-TAK1 (H) was measured using ImageJ. The results are presented as means ± standard deviation (SD, n = 3 independent experiments). \(^{\blacklozenge }\)P < 0.05. I and J. Ctrl A549 and FFAR2KO A549 (I) or Ctrl H1299 and FFAR2KO H1299 (J) cells were treated with vehicle, HKLM, and poly(I:C), as indicated, and the NF-κB luciferase reporter assay was performed. The results are presented as means ± standard deviation (SD, n = 3 independent experiments). **P < 0.01, and ***P < 0.001. K and L. Ctrl A549 and FFAR2KO A549 (K) or Ctrl H1299 and FFAR2KO H1299 (L) cells were treated with vehicle, HKLM, and poly(I:C), as indicated, and cAMP levels were measured. The results are presented as means ± standard deviation (SD, n = 3 independent experiments). ***P < 0.001