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. 2013 Aug 14;8(8):e72488. doi: 10.1371/journal.pone.0072488

Figure 2. Gal-9 differentially regulates IL-12 and IL-23 gene transcriptions in THP-1 cells through STAT-3 signaling.

Figure 2

A) Western blot detection of Gal-9 and phosphorylation of STAT-1 proteins in THP-1 cells transfected with Gal-9 plasmid or siRNA. THP-1 cells were transfected with either pBKCMV3-Gal-9 plasmid (Gal-9) or pBKCMV3 empty vector (Con), or Gal-9 silencing siRNA or control siRNA. After 24~48 h transfection, the cells were subjected to Western blot analysis of Gal-9 and pSTAT-1 proteins. β-actin or total STAT-1 served as loading control to normalize target gene levels. Data are shown as representative imaging (left) and corrected optimal densitometry (O.D.) values from three independent experiments for STAT-1 (right). NS = no significance. B) Western blot detection of pSTAT-3 protein in THP-1 cells transfected with Gal-9 plasmid in the presence or absence of TLR stimulations. THP-1 cells were transfected with either Gal-9 or Control plasmid for 24 h, stimulated with or without LPS/R848 for 6 h, followed by Western blot analysis of pSTAT-3 protein. Total STAT-3 served as loading control to normalize target gene levels. Representative imaging and corrected O.D. values from three independent experiments are shown. *P<0.05. C) Western blot detection of pSTAT-3 protein in THP-1 cells transfected with Gal-9 siRNAs with or without 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 Western blot analysis of pSTAT-3 protein. Total STAT-3 served as loading control. Representative results and corrected O.D. values from three experiments are shown. *P<0.05, NS = no significance. D) Luciferase assay for Tim-3, IL-12, IL-23 promoter activities in Gal-9-transfected THP-1 cells in the presence of STAT-3 inhibitor. pBKCMV3-Gal-9 or control plasmid was transfected into THP-1 cells, along with Tim-3, IL-12p35, IL-23p19, or IL-12/IL-23p40 promoter reporter vectors, in the presence of STAT3-specific inhibitor or DMSO for 24 h, followed by luciferase assays. Data are mean ± SE of triplicate samples for relative luciferase units (RLU). *P<0.05, **P<0.01, ***P<0.001, NS = no significance. E) Intracellular Gal-9 and TLR stimulation on Tim-3, IL-12, IL-23 promoter activities in THP-1 cells treated with STAT-3 inhibitor and TLR stimulation. pBKCMV3-Gal-9 or control vector was transfected into THP-1 cells, along with Tim-3, IL-12p35, IL-23p19, or IL-12/IL-23p40 promoter reporter vectors, in the presence of STAT3-specific inhibitor for 24 h. The cells were stimulated without or with LPS/R848 for 6 h, followed by luciferase assays as described in Methods. Data are mean ± SE of triplicate samples for relative luciferase units (RLU). *P<0.05, **P<0.01, ***P<0.001, NS = no significance.