Figure 4: FYT21 inhibits TLR4 dimerization induced by LPS.

THP1 cells were incubated o/n with a range of FYT21 without (a) or with (b) 100 ng ml⁻¹ of LPS, and the expression of CD14, CD282 (TLR2) and CD284 (TLR4) was measured on a FACSCalibur. Results, expressed as mean fluorescence intensity (MFI), are means±s.e.m. of eight experiments. (c) The expression of monomeric TLR4 on RAW-Blue cells, as compared with dimerized/LPS-bound TLR4 (n=4), using an antibody that only binds to the monomeric form. A representative FACS histogram is shown to the right. Results of control samples were set at 100%. The effect of 10 μM FYT21 or 50 μg ml⁻¹ of polymyxin B on LPS-induced TLR4 activation are expressed as a percentage relative to the control. Results are means±s.e.m. of four experiments. Values are significantly (*P<0.05 and ***P<0.0005) different from the controls as analysed using a one-way ANOVA with a Dunnett’s multiple comparisons test. (d) Cells were incubated with LPS and/or FYT21 for 30–60 min followed by processing and Au-labelling with antibodies against TLR4, LPS and FYT21: (I) monomeric TLR4, (II) LPS-induced dimerization of TLR4, (III) binding of FYT21 to the membrane, (IV) the interaction between FYT21, LPS and TLR4 when the cells were preincubated for 30 min with FYT21 or (V) LPS, or when added simultaneously (VI; scale bar, 200 nm).