Journal: PLoS ONE

Article Title: Human MiR-544a Modulates SELK Expression in Hepatocarcinoma Cell Lines

doi: 10.1371/journal.pone.0156908

Figure Lengend Snippet: (A) The potential targets of indicated miRNAs were cloned in the reporter vector psiCheck2. One construct was used for both miR-181a and miR-181b (miR-181a/b), since their target sequences resulted overlapped. 48h after transfection, luciferase activities were recorded; the Renilla luciferase activity (Rl) was normalized to the firefly luciferase activity (luc) and the uninhibited activity relative to the parental vector psiCheck2 (V) was set to 1. (B) Expression of miRNAs were evaluated by qRT-PCR and reported as arbitrary units (A.U.) (C) Following the same procedure as above (A), a control construct (I), containing the inverted sequence targeted by miR-544a, was tested. P-values < 0.01 were evaluated by Student’s t-test and indicated with **.

Article Snippet: HepG2 and HuH-7 cells were transfected with 0.2 and 0.05 μg of reporter constructs, respectively; miScript miR-544a mimic, miScript miR-544a inhibitor, and their controls with unrelated sequences (AllStars Negative Control and miScript Inhibitor Negative Control, respectively; all from Qiagen) were transfected at 50nM along with 50ng of phRL-tk plasmid encoding Renilla luciferase to monitor transfection efficiency.

Techniques: Clone Assay, Plasmid Preparation, Construct, Transfection, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR, Control, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Notch activation promotes endothelial quiescence by repressing MYC expression via miR-218

doi: 10.1016/j.omtn.2021.07.023

Figure Lengend Snippet: Notch activation upregulates miR-218 and its host gene Slit2 in ECs (A) HUVECs were transfected with AdNIC or AdCtrl adenovirus for 48 h. Hey1, Slit2, and miR-218 RNA levels were determined by quantitative real-time PCR (n = 5). (B) HUVECs were cultured in the presence of DAPT (25 μM) or DMSO for 48 h, and Hey1, Slit2, and miR-218 RNA levels were determined by quantitative real-time PCR (n = 4). (C) HUVECs were cultured with dishes coated with Dll4 or PBS for 48 h. Hey1, Slit2, and miR-218 RNA levels were evaluated by quantitative real-time PCR (n = 3). (D) HUVECs were cultured as in (C) in the presence or absence of DAPT for 48 h. Hey1, Slit2, and miR-218 RNA levels were determined by quantitative real-time PCR (n = 4). (E) Reporter assay. A representative illustration of the structure of Slit2 gene and the reporter is shown on the left. Luciferase activity was determined and calibrated in HEK293T cells transfected with pGL3-Slit2 (100 ng), phRL-TK (5 ng), and increasing amount of pEFBOS-NIC (0, 50, 100 ng) for 48 h (n = 8). Error bars, means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: In other cases, HUVECs were co-transfected with 100 ng pGL-MycUTR5, pGL-MycUTR3, pGL-hnRNPAUTR3, pGL-EYAUTR3, together with phRL-TK (5 ng) and pGV317-miR-218 (0, 50, 100 ng, Genechem).

Techniques: Activation Assay, Transfection, Real-time Polymerase Chain Reaction, Cell Culture, Reporter Assay, Luciferase, Activity Assay