RND3 Potentiates Proinflammatory Activation through NOTCH Signaling in Activated Macrophages

Figure 4

RND3 modulates NFκB transcriptional activity. (a) Analysis of NF-κB transcriptional activity in Raw 264.7 cells activated with LPS (100 ng/ml) and IFN-γ (10 U/ml) and transiently transfected with a NF-κB luciferase reporter (NFκB–LUC) and a Rnd3 expression vector (RND3) or its corresponding control vector (VV) (left panel), or Rnd3-specific shRNA (shRND3) or unspecific shRNA control vector (VV) (right panel). 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 corresponding control, treated cells. (b) Analysis of NF-κB transcriptional activity in Raw 264.7 cells activated with LPS (100 ng/ml) and IFN-γ (10 U/ml) and transiently transfected with a NF-κB luciferase reporter (NFκB–LUC) and a Rnd3 expression vector (RND3), or Notch1-specific shRNA (shN1) or both. 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 corresponding control, treated cells. (c) qPCR analysis of Tnf-α, IL-6, and Cox-2 mRNA expression or (d) analysis of secreted TNFα and IL-6 by ELISA in peritoneal murine macrophages transfected with Rnd3-specific siRNA (siRND3) or control siRNA (siC) and activated for 6 hr with LPS (100 ng/ml) and IFN-γ (10 U/ml). All data are referred to stimulated macrophages treated with siControl RNA set to 100. qPCR was performed in triplicate with riboprotein P0 as the internal control. Means ± SD of three independent experiments are shown. Student unpaired t-test was used for statistical analyses between two groups at the level of , compared to control, untreated cells. (e) Western blot analysis of Rel-B in Raw 264.7 cells transiently transfected with a control (Raw) or Rnd3 expression vector (Raw-RND3) (left panel); and in peritoneal murine macrophages transfected with Rnd3-specific siRNA (siRND3) or control siRNA (siC) (right panel), activated with LPS (100 ng/ml) and IFN-γ (10 U/ml). ERK-2 expression was used as a loading reference. A representative image of at least three experiments is shown. 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 the corresponding empty vector, untreated cells.

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