RND3 Potentiates Proinflammatory Activation through NOTCH Signaling in Activated Macrophages

Figure 2

RND3 modulates NOTCH signaling in activated macrophages. (a) Analysis of NOTCH transcriptional activity in control and activated Raw 264.7 cells transiently transfected with a CBF luciferase reporter gene (CBF–LUC) in the presence of a RND3 expression vector (RND3) or its corresponding control vector (VV) (left panel) or Rnd3-specific shRNAs (shRND3) or its corresponding unspecific shRNA control vector (VV) (right panel). Means ± SD of four independent experiments are shown. One-way ANOVA analysis with Bonferroni’s post-tests was performed. Statistical significance was determined at the level of , compared to control untreated cells; ^### compared to the corresponding control vector, treated cells. (b) Analysis of NOTCH transcriptional activity in control and activated Raw 264.7 cells transiently transfected with a CBF luciferase reporter gene (CBF–LUC) in the presence of a RND3 expression vector (RND3) or an intracellular active domain of NOTCH1 expression vector (NIC1) or both, and their corresponding controls vector (VV). Means ± SD of three independent experiments are shown. One-way ANOVA analysis with Bonferroni’s post-tests was performed. Statistical significance was determined at the level of , compared to control untreated cells; ^### compared to the control vector, treated cells. (c) Analysis of NOTCH transcriptional activity in control and activated Raw 264.7 cells transiently transfected with a CBF luciferase reporter gene (CBF–LUC) in the presence of a RND3 expression vector (RND3) or Notch1-specific shRNA vector (shN1) or both, and their corresponding control vectors. Means ± SD of three independent experiments are shown. One-way ANOVA analysis with Bonferroni’s post-tests was performed. Statistical significance was determined at the level of , , compared to control untreated cells; ^##, compared to the control vector, treated cells; ^§§§ compared to Rnd3-transfected, treated cells. (d) Analysis of NOTCH transcriptional activity in control and activated Raw 264.7 cells transiently transfected with a CBF luciferase reporter gene (CBF–LUC) in the presence of an active intracellular domain of NOTCH1 expression vector (NIC1) or Rnd3-specific shRNA (shRND3) or both, and their respective control vectors. Means ± SD of four independent experiments are shown. One-way ANOVA analysis with Bonferroni’s post-tests was performed. Statistical significance was determined at the level of, , , compared to control untreated cells; ^#, ^### compared to the control vector, treated cells; ^§§§ compared with cells transfected with intracellular active domain of NOTCH1 expression vector and control vector. Cells were stimulated with LPS (100 ng/ml) and IFN-γ (10 U/ml) for 24 hr before analysis, 1 day after transfection. pRLTK was used as an internal control vector for transfection and normalized luciferase/Renilla values are represented.

(a)

(b)

(c)

(d)