Figure 3.

The involvement of MyD88 and IRAK4 in LPS-induced increase in Caco-2 permeability. (A). siRNA MyD88 transfection resulted in a near-complete depletion of MyD88 expression as assessed by Western blot analysis and relative densitometry. The Western blot analysis was performed 72 h after siRNA MyD88 transfection. siRNA-induced silencing of MyD88 prevented LPS-induced increase in Caco-2 inulin flux (B) and drop in TER (C). The mean TER for control Caco-2 monolayers was 536 ± 20 Ω • cm². (D). Effect of LPS on phospho-IRAK4 expression assessed by Western blot. LPS (0.3 ng/ml) treatment induced a time-dependent increase in phospho-IRAK4 expression. The densitometry analysis indicates a time-dependent increase in relative levels of phoshpo-IRAK4 after LPS treatment and no significant change in the total IRAK4 levels. (E). Effect of LPS on IRAK4 activity measured by IRAK4 Kinase Enzyme System and ADP-Glo Kinase Assay. (F). Effect of IRAK4 inhibitor (N-(2-Morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole, 200 nM) on Caco-2 IRAK4 activity. IRAK4 inhibitor prevented the LPS-induced increase in Caco-2 IRAK4 activity. (G). Effect of IRAK4 inhibitor on Caco-2 TER. IRAK4 inhibitor prevented the LPS-induced drop in Caco-2 TER. The mean TER for control Caco-2 monolayers was 516 ± 9 Ω • cm². (H) Effect of siRNA-induced silencing of MyD88 on IRAK4 activity in Caco-2 monolayers. n=4. *, p<0.0001 vs control. **, p<0.0001 vs. LPS treatment.