Fig 1: The NF‐κB response reporter gene (Promega's pNL3.2.NF‐kB NlucP/Nf‐kB/hygro) was transfected into hCMs to identify Nfκβ gene activity. (A) As a control, Tnfa was added to hCMs, resulting in a significant elevation in luciferase activity. (B) Incubating hCMs in Ang2/PE for 3 h resulted in a significant increase in NF‐κB response. This increase was prevented if hCMs were subjected to CTP‐miRNA106a prior to the addition of Ang2/PE. C‐D) One downstream gene activated by NF‐κB is IL‐1b. Western analyses showed significant increases in IL‐1b beginning at 24 h through 144 h of Ang2/PE incubation. Here, CTP‐miRNA106a was added to hCMs 72 h after Ang2/PE incubation (Rescue), resulting in a significant reduction in IL‐1b protein expression N = 21 (#p < .001 for all timepoints except untreated. & = No significant difference compared with untreated). Protein expression in CTP‐miRNA106a‐treated cells was equivalent to untreated hCMs (i.e., rescued). (E) The Media from hCMs treated for 24 h with Ang2/PE and hCMs pre‐treated with CTP‐miRNA106a, followed by Ang2/PE for 24 h, were compared with media from untreated hCMs using a sensitive ELISA assay. The secretion of IL‐1b produced in hCMs by Ang2/PE was suppressed in CTP‐miRNA106a + Ang2/PE‐treated hCMs. The blank is to show no background from the plate and media. (F) FACs’ analyses of hCMs treated with brefeldin A for the last 6 h of a 24 h Ang2/PE incubation further confirmed the increased expression of IL‐1b (blue bell curve). The upper right panel shows a clear shift of cells (red curve) back towards untreated levels if incubated in CTP‐miRNA106a prior to Ang2/PE. The lower left panel shows the same shift of hCMs (red curve) back towards baseline if hCMs are treated with Ang2/PE first for 24 h, followed by the addition of CTP‐miRNA106a for at least 24 h. As a control, miRNA106a (no CTP) was transfected into hCMs using a lipid‐based transfection reagent, followed by Ang2/PE. The results show that the shift in cells (red curve) back to baseline was caused by miRNA106a. N = 3 for Western 0–144 timepoints. N = 21 for CTP‐miRNA106a rescued samples. One‐way ANOVA identified significance, followed by unpaired t‐tests comparing two variables at a time to identify significance.