The siRNA range of 0.05–0.5 μM was found to efficiently reduce ER-α mRNA expression. We used the 0.5 μM dose of siRNA for further experiments. The role of ER-α in TLR2 agonist-induced MCP1 Z-VAD-FMK clinical trial production was determined by using the ER-α inhibitor MPP at different doses in the presence and absence of TLR2 agonist in vitro. The cells were incubated with MPP 8 h before LTA treatment. After 24 h, supernatants were collected to assay MCP1 production. The

results presented in Fig. 5A demonstrated that MPP at the doses of 1.6–3.2 μM has the ability to decrease MCP1 production significantly (p <0.005) in TLR2 agonist Pam3CsK4-treated mesangial cells compared to cells treated with Pam3CsK4 without MPP. The untreated cell supernatants were used as the control for MCP1 production. The ER-α siRNA- and scrambled siRNA-transfected mesangial cells were treated in vitro with Pam3CsK4 (10 ng/ml) for a period of 24 h. After that, supernatants were collected, and MCP1 production was estimated by ELISA. The results presented in Fig. 5B demonstrated that mesangial cells transfected with scrambled siRNA had the ability to produce MCP1 following

incubation with Pam3CsK4 compared to un-stimulated controls. The ER-α siRNA-transfected mesangial cells demonstrated significantly reduced MCP1 production following Pam3CsK4 treatment in vitro compared to control (scrambled) siRNA-transfected cells (p <0.005). Mesangial cells transfected http://www.selleckchem.com/products/OSI-906.html with either ER-α siRNA or scrambled siRNA without Pam3CsK4 treatment were used as controls for MCP1 production. Next, we determined the relative mRNA expression of MCP1

in the ER-α siRNA-transfected mesangial cells after incubation with Pam3CsK4. The mRNA expression of MCP1 in ER-α siRNA-transfected mesangial cells was found to decrease significantly (p <0.005) compared to scrambled siRNA-transfected Pam3CsK4-treated mesangial cells ( Fig. 5C). Next, we determined the effect of estrogen on MCP1 production in TLR2 ligand-treated mesangial cells. For this, mesangial cells (5×105) were incubated with estrogen (10 nM), LTA (10 ng/ml) and the combination of estrogen and LTA for 24 h. After that, supernatants were collected to assay for MCP1 using ELISA. The results presented in Fig. 6 demonstrated that the TLR2 ligand PRKD3 lipoteichoic acid induced production of MCP1 similar to that observed with Pam3CsK4-treated mesangial cells in vitro. Treatment with estrogen was found to inhibit LTA-induced MCP1 production in mesangial cells (p <0.05). However, treatment with estrogen alone with or without ER-α siRNA transfection had no effect on MCP1 production. Additionally, treatment of ER-α siRNA-transfected mesangial cells with estrogen had no effect on MCP1 production. The untreated mesangial cells and scrambled siRNA transfected cells were used as controls for MCP1 production in vitro. Mesangial cells are known to play a vital role in kidney inflammation [1].