Fig 1: Sulfur compounds inhibit the expression of TLRs. (A) The expression levels of TLR2 and TLR4 mRNA in THP-1 cells were detected by real-time PCR after treatment with 20 mM glucose and NTS (2 and 4 µg/mL) for 24 h and normalized to GAPDH mRNA. The values are presented as means ± SEM of three independent experiments performed in duplicate (n = 3). *** p < 0.001 (ANOVA test). # The mean difference is significant at the 0.001 level. (B) Western blot analysis of THP-1 cells treated with 2 and 4 µg/mL NTS for 24 h showing the inhibition of high glucose-induced TLR2 and TLR4 expression. (C) The representative expression levels of proteins following NTS treatment were determined by densitometry and normalized to ß-actin. The values are presented as means ± SEM of three independent experiments performed in duplicate (n = 3). *** p < 0.001 (ANOVA test). # The mean difference is significant at the 0.001 level. (D) Real-time PCR data of mRNA after treatment with 20 mM glucose and MSM (100 and 200 mM) for 24 h showing the relative expression levels of TLR2 and TLR4 normalized to GAPDH mRNA. The values are presented as means ± SEM of three independent experiments performed in duplicate (n = 3). *** p < 0.001 (ANOVA test). # The mean difference is significant at the 0.001 level. (E) Western blotting analysis of THP-1 cells treated with 100 and 200 mM MSM for 24 h showing the inhibition of high glucose-induced TLR2 and TLR4 expression. (F) The representative expression levels of proteins following MSM treatment were determined by densitometry and normalized to ß-actin. The values are presented as means ± SEM of three independent experiments performed in duplicate (n = 3). *** p < 0.001 (ANOVA test). # The mean difference is significant at the 0.001 level. (G) Flow cytometry analysis showing the inhibition of the high glucose-induced expression of TLR2 and TLR4 by 4 µg/mL NTS and 200 mM MSM. Mannitol and 5.5 mM glucose were used as negative controls for high glucose-induced inflammatory reaction. Non-staining is the control not stained with anti-TLR2 and 4 antibodies for flow cytometry.