epscm with or without ifnγ stimulation  (STEMCELL Technologies Inc)

Journal: PNAS Nexus

Article Title: Vascular endothelial cell-specific disruption of the profilin1 gene leads to severe multiorgan pathology and inflammation causing mortality

doi: 10.1093/pnasnexus/pgad305

Figure Lengend Snippet: Effect of endothelial Pfn1 depletion on immunomodulatory cytokine/chemokine secretion in vitro and in vivo. A and B) Relative abundance of indicated cytokines/chemokines circulating in the serum of Pfn1 WT vs Pfn1 EC-KO mice collected either 8–10 days (mid-stage, panel A) or 15–18 days (late-stage, panel B) after the last TMX administration. Data representative of at least three separate litters with “ n ” representing the total number of mice in each group pooled from different litters. C and D) Real-time quantitative RT-PCR ( panel C ; n = 3 experiments) and immunoblot ( panel D ) validations of Pfn1 depletion in Ad-Cre–infected EC with Ad-GFP–infected cells serving as control (18S RT-PCR and GAPDH blots serve as the housekeeping gene control and loading control for RT-PCR and immunoblot experiments, respectively). E) Relative abundance of indicated cytokines/chemokines in the CM of Ad-Cre–infected EC relative to Ad-GFP–infected control cells (data represent the average fold change based on three independent experiments). F) Relative abundance of IL6 mRNA expression in circulating primary F4/80+ cells isolated from mice with the indicated genotypes ( n = 6 mice/group pooled from two separate litters). G) Relative abundance of IL6 mRNA expression in macrophages differentiated from primary BMDM and exposed to the CM of Ad-Cre– vs Ad-GFP–infected EC, either in the absence or in the presence of IFNγ stimulation (data summarized from five independent experiments). * P < 0.05 and ** P < 0.01.

Article Snippet: After 7 days, macrophages were replated and exposed to the CM derived from EC with or without additional stimulation of 100 ng/ml IFNγ (PeproTech, Cranbury NJ) for 24 h.

Techniques: In Vitro, In Vivo, Quantitative RT-PCR, Western Blot, Infection, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation