LPS induces Irf1-mediated Lif transactivation in lung endothelial cells
(A) Venn analysis identifying profoundly up-regulated Irf1 target genes in murine lung endothelial cells (MLECs). (B) Web-based term association analysis to identify which Irf1 target gene(s) have the strongest association with endothelial regeneration-associated search terms. (C) qPCR analysis of Irf1 and Lif gene expression in MLECs isolated from mice pre- and post-LPS. (D) Representative immunoblots and densitometric quantification of Irf1 and Lif protein levels in MLECs pre- and post-LPS challenge. (E) qPCR analysis of IRF1 and LIF gene expression in human lung microvascular endothelial cells (HLMVECs) isolated from mice pre- and post-LPS. (F) Representative immunoblots and densitometric quantification of IRF1 and LIF protein levels in HLMVECs pre- and post-LPS challenge. (G) qPCR analysis of IRF1 and LIF gene expression in human lung microvascular endothelial cells (HLMVECs) transfected with control or IRF1 vector. (H) Representative immunoblots and densitometric quantification of IRF1 and LIF protein levels in HLMVECs with control vector or IRF1 overexpression. (I) Schematic of the human LIF promoter region depicting the highly-conserved IRF1 ISRE binding site at −102 ∼ −127 bp. The WT and MUT ISRE sequences used in panel (K) are provided. (J) HLMVECs transduced with either control or IRF1 plasmids, subjected to vehicle or LPS conditions for 8 h, followed by ChIP-qPCR assays for detection of IRF1 binding to the −102 bp binding site within the LIF promoter region. (K) HLMVECs co-transfected with control or IRF1 plasmid along with one of three luciferase (Luc) reporter gene constructs. Schematic representations of Luc constructs are indicated. All experiments: n = 6 mice or six independent biological replicates per group. Data represented as means ± SDs. **P<0.01 [one-way ANOVA (C) and two-way ANOVA (E–H) with Bonferroni post-hoc tests].