RND3 Potentiates Proinflammatory Activation through NOTCH Signaling in Activated Macrophages

Figure 5

RND3 modulates STAT1 transcriptional activity in activated macrophages. (a) Analysis of STAT1 transcriptional activity in Raw 264.7 cells activated with LPS (100 ng/ml) and IFN-γ (10 U/ml) and transiently transfected with a STAT1 luciferase reporter (STAT1–LUC) and a Rnd3 expression vector (RND3) or its corresponding control vector (VV) (left panel), or Rnd3-specific shRNAs (shRND3) or unspecific control shRNA 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) qPCR analysis of Rnd3 and Irf-1 mRNA expression (left panel), or western blot analysis of RND3 or IRF1 (right panel) 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). qPCR data are referred to control stimulated macrophages 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. (c) qPCR analysis of Rnd3 and Irf-1 mRNA expression (left panel), or Western blot analysis of RND3 or IRF1 (right panel) in Raw 264.7 cells transiently transfected with a control (Raw) or RND3 expression vector (Raw-RND3) 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 independent experiments is shown in Western blot analysis. One-way ANOVA analysis with Bonferroni’s post-tests was performed. Statistical significance was determined at the level of , , compared to control untreated cells. (d) qPCR analysis of Cxcl10 mRNA expression 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). qPCR data are referred to control stimulated macrophages 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.

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