Figure 4

TIGFs lack TLR2 expression and fail to respond to TLR2 agonists. (a) Western blot analysis of TLR2 protein expression in PHGF and TIGF total cell lysates. U-251 MG cells overexpressing hTLR2 were used as a positive control and actin was used as a loading control. Results presented are representative of two independent experiments and full-length blots are presented in Supplementary Fig. S2. (b) Relative mRNA expression of TLR2 in PHGFs and TIGFs (n = 3) that were left unstimulated (med) or were stimulated with TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 24 h. (c) Relative mRNA expression of IL6, IL8 and COX2 in PHGFs and TIGFs stimulated with Pam2CSK4 (1 μg/ml) or Pam3CSK4 (1 μg/ml) for 24 h. Data for PHGFs from individual donors (n = 3) and TIGFs from independent experiments (n = 3) are shown on a heat map as column Z-scores calculated from ΔCt values relative to a housekeeping gene (RPLP0). (d) Secretion of IL-6 and IL-8 by PHGFs and TIGFs (n = 3) and stimulated as in (c). (e) TLR2 promoter methylation in PHGFs and TIGFs (n = 3) analyzed using the EpiTect methyl II PCR assay and presented as mean percentage of methylated DNA + SEM. (f) Relative TLR4 mRNA expression in PHGFs and TIGFs (n = 3). (g) Western blot analysis of TLR4 protein expression in total cell lysates of PHGFs and TIGFs. Actin was used as a loading control. (h) Secretion of IL-6 and IL-8 by PHGFs and TIGFs (n = 3–4) infected with increasing MOI of F. nucleatum (10, 50) for 24 h. (i) Secretion of IL-6 and IL-8 by PHGFs and two independent batches of TIGFs (TIGF-1 and TIGF-2) (n = 2–3) infected with increasing MOI of P. gingivalis (10, 50, 100). Data are shown as mean relative mRNA expression + SEM (b,f) or mean concentration + SEM (d,h,i). ***P < 0.001; 1-way ANOVA followed by Bonferroni multiple comparison test.