Fig. 5. Endocytosis of palmitate/TLR4–MD2 complex generates NOX2-mediated ROS.

a RAW 264.7 cells were stimulated with 200 μM palmitate for 1 h. Total cell lysates, membrane fractions or cytosol fractions of RAW 264.7 cells were subjected to Western blotting or immunoprecipitation (IP). With anti-NOX2, immunocomplexes from cytosolic fractions were followed by immunoblot analysis with antibody of TLR4. b, c BMDMs of WT and Nox2 KO mice were stimulated with 200 μM palmitate for 1 h and then they were subjected to Western blotting and qRT–PCR analyses, respectively. d RAW 264.7 macrophages were subjected to FACS analyses to measure ROS generation after each siRNA silencing. To inhibit CD36-mediated palmitate uptake, RAW 264.7 cells were pre-treated with sulfosuccinimidyl oleate (SSO) for 10 min. e Direct perfusion of the liver with palmitate. C-16 BODIPY (10 μM) and palmitate (500 μM) were infused via the portal vein for 10 minutes. Both the superior and inferior vena cava were clipped for the last one minute to trap the solution in the liver. f, g After perfusing C16-BODIPY or palmitate through the liver of WT, Tlr4 KO, Nox2 KO and Myd88/Trif double KO mice, successful delivery of C16-BODIPY and ROS generation were assessed in freshly isolated CD11b⁺F4/80^low macrophages, CD11b⁺F4/80^high Kupffer cells and CD11b⁺Ly6G^high neutrophils, respectively. Data are representative of three independent experiments using isolated liver immune cells from 3 (f, g) mice per group. Data are expressed as the mean ± s.e.m. and analyzed by Student’s t-test, *P < 0.05, **P < 0.01 in comparison with the corresponding controls