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STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis

Figure 3

Effect of STAT1 dependent signal integration on chemokine expression.

WT and STAT1−/− VSMCs, HMECs or WT aortic ring segments were treated as described in Fig. 1. A, RNA from VSMCs was isolated and qRT-PCR for Ccl5, Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10, Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p<0.05 was considered as significant. Data were tested for significance by one-way ANOVA followed by post-hoc Tukey or unpaired two-tailed student T-test when appropriate. E, ChIP-qPCR analysis of the Cxcl10 promoter region containing NFκB and ISRE binding sites show the enrichment with STAT1, NFκB and IRF1 antibodies compared with IgG control in an IFNγ, LPS or IFNy+LPS-dependent manner in WT VSMCs. Immunoprecipitated DNA was quantified by qPCR and normalized to values obtained after amplification of unprecipitated (input) DNA. A representative experiment is shown.

Figure 3

doi: https://doi.org/10.1371/journal.pone.0113318.g003