A) RT-PCR detection of Tim-3, IL-12p35, IL-23p19, and IL-12/IL-23p40 mRNAs in THP-1 cells transfected with Gal-9 plasmid in the absence or presence of TLR stimulations. THP-1 cells were transfected with either pBKCMV3-Gal-9 plasmid (Gal-9) or pBKCMV3 empty vector (Con) for 24 h, stimulated with or without LPS and R848 for 6 h, followed by RT-PCR measuring Tim-3, IL-12p35, IL-23p19, IL-12/IL-23p40 mRNA expressions. β-actin served as loading control to normalize target gene levels. Data are shown as representative imaging (left) and mean ± SE of corrected optimal densitometry (O.D) values from three independent experiments (right). *P<0.05, **P<0.01, ***P<0.001, NS = no significance, analyzed by multiple comparisons testing/least significant difference on the ANOVA Prism software. B) RT-PCR detection of Tim-3, IL-12p35, IL-23p19, and IL-12/IL-23p40 mRNAs in THP-1 cells transfected with Gal-9 siRNAs in the absence or presence of TLR stimulations. THP-1 cells were transfected with either Gal-9 or control siRNAs for 48 h, stimulated with or without LPS/R848 for 6 h, followed by RT-PCR measuring Tim-3, IL-12p35, IL-23p19, IL-12/IL-23p40 mRNA expressions. β-actin served as loading control. Data are shown as representative imaging (left) and mean ± SE of corrected optimal densitometry (O.D) values from three independent experiments (right). *P<0.05, **P<0.01, ***P<0.001, NS = no significance, analyzed by multiple comparisons testing/least significant difference on the ANOVA Prism software. C) Enhanced Gal-9 expression and TLR stimulation on Tim-3, IL-12, IL-23 promoter activities. pBKCMV3-Gal-9 or control vector was transiently transfected into THP-1 cells, along with either Tim-3, or IL-12p35, or IL-23p19, or IL-12/IL-23p40 promoter luciferase reporter vectors. 24 h after transfection, the cells were stimulated with or without LPS/R848 for 6 h, followed by luciferase assays for reporter gene transcriptional activities as described in Methods. Data are shown as mean ± SE of triplicate samples for relative luciferase units (RLU). *P<0.05, **P<0.01, ***P<0.001, NS = no significance, analyzed by multiple comparisons testing/least significant difference on the ANOVA Prism software. D) Silenced Gal-9 expression and TLR stimulation on Tim-3, IL-12, IL-23 promoter activities. Gal-9 or control siRNAs was transiently transfected into THP-1 cells, along with either Tim-3, or IL-12p35, or IL-23p19, or IL-12/IL-23p40 promoter reporter vectors. 48 h after transfections, the cells were stimulated with or without LPS/R848 for 6 h, followed by luciferase assays. Data are shown as mean ± SE of triplicate samples for relative luciferase units (RLU). *P<0.05, **P<0.01, ***P<0.001, NS = no significance, analyzed by multiple comparisons testing/least significant difference on the ANOVA Prism software.