TLR9 and RIG-I Signaling in Human Endocervical Epithelial Cells Modulates Inflammatory Responses of Macrophages and Dendritic Cells In Vitro
Figure 1
Endocervical cells (End1/E6E7) express TLR9 and RIG-I.
A. RT-PCR analysis of TLR9 and RIG-I gene expression. (a & b). End1/E6E7 cells ells were seeded at a density of 1×105 cells/well in a 24-well plate and treated for 6 hrs with TLR9 and RIG-I ligands (10 µg/ml). RT-PCR results show that expression of TLR9 and RIG-I mRNAs was significantly upregulated in End1/E6E7 cells upon stimulation with TLR9 and RIG-I ligands, CpG-ODN and poly (I: C)LL respectively compared to medium controls. (c) & (d) A quantitative densitometry analysis of TLR9 (c) and RIG-I (d) expression in End1/E6E7 cells. Values were calculated as the mean (± SD) of three separate experiments performed on different days. Level of significance (**p<0.01; ***p<0.01) was calculated by unpaired t test. B. Analysis of TLR9 and RIG-I mRNA in End1/E6E7 cells by qPCR. End1/E6E7 cells (1×105) were stimulated with ligands of TLR9 (CpG-ODN: 10 µg/ml) and RIG-I {poly (I: C) LL, 10 µg/ml} for 6 hrs. Values represent mean (±SD) of three experiments performed in duplicates on different days (p<** 0.01; ***p<0.001) were calculated by ANOVA test followed by Bonferroni analysis. C. Flow cytometry analysis of TLR9 and RIG-I expression. Flow cytometry was used to compare the percentage of permeabilized End1/E6E7 cells labeled by anti-TLR9 and anti-RIG-I antibodies incubated with media (a, c) and after stimulation with their cognate ligands, CpG-ODN and Poly (I: C)LL (b, d) respectively. Comparison of TLR9 and RIG-I positivity was made by comparing fluorescence in the M2 channel. The figures shown are the representative pictures from three independent experiments. (FLI: log fluorescence intensity). D. Confocal images showing the expression of TLR9 and RIG-I in End1/E6E7 cells. Figures (a–f) End1/E6E7 cells were treated with media or CpG-ODN for 6 hrs and subsequently stained with anti-TLR9 antibody. Figures (g–l) End1/E6E7 cells were treated with media or Poly(I:C)LL for 6 hrs and subsequently stained with anti-RIG-I antibody. The figures shown are the representative picture from three independent experiments. (Magnification X 63) (Scale 10 µm).