Journal: Medicina

Article Title: Inhibiting MiR-33a-3p Expression Fails to Enhance ApoAI-Mediated Cholesterol Efflux in Pro-Inflammatory Endothelial Cells

doi: 10.3390/medicina61020329

Figure Lengend Snippet: Challenging iMAECs with LPS induces VCAM-1 protein expression and leads to increased miR-33a-3p expression. ( A , B ). Representatives immunoblot ( A ) and densitometry ( B ) of vehicle (Veh)-treated and LPS-challenged iMAECs for measuring VCAM-1 protein with GAPDH used as the housekeeping protein. Data points ( B ) are from two independent experiments with five biological replicates used in each experiment. ( C ) End-point RT-PCR and restriction digest of PCR products assessed by TBE agarose gel electrophoresis to detect miR-33a-3p. MiR-33a-3p cDNA contains one Tsp RI restriction site but lacks Bsr DI restriction sites. M, DNA marker; NTC, non-template control PCR reaction; minus (−) indicates non-digested amplicons; T, Tsp RI-digested amplicons; B, Bsr DI-digested amplicons; pMAEC total RNA was used as template for positive technical control end-point RT-PCR and restriction digestion reactions. ( D ) MiR-33a-3p expression measured in vehicle-treated and LPS-challenged iMAECs via RT-qPCR. Data points show two independent experiments with six biological replicates used for both experiments. ( A ) Size markers are in kDa; ( B , D ) bars are group means. AU = arbitrary units.

Article Snippet: To measure mRNA expression of ABCA1 and Niemann–Pick type C1 (NPC1), we first converted total RNA extracted from cultured iMAECs into cDNA via using a qScript cDNA Ultra SuperMix Kit (Quantabio, Beverly, MA, USA) [ ].

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Control, Quantitative RT-PCR

Journal: Medicina

Article Title: Inhibiting MiR-33a-3p Expression Fails to Enhance ApoAI-Mediated Cholesterol Efflux in Pro-Inflammatory Endothelial Cells

doi: 10.3390/medicina61020329

Figure Lengend Snippet: Transfecting pro-inflammatory endothelial cells with pA3p decreases miR-33a-3p expression. ( A ) End-point RT-PCR coupled with TBE agarose gel electrophoresis was used for detecting transgenic anti-miRs within LPS-challenged iMAECs transfected with either pScr of pA3p. Top labels: U6, positive control PCR reactions to detect U6; Scr, PCR reactions for detecting scrambled anti-miR; A3p, PCR reactions for detecting anti-miR-33a-3p. Bottom labels: Scr, cDNA from pScr-transfected iMAECs; A3p, cDNA from pA3p-transfected iMAECs; NTC, non-template control. ( B , C ) Percent of intact GFP + cells detected via flow cytometry counted from LPS-challenged iMAECs transfected with either pScr ( B ) or pA3p ( C ) versus respective mock-transfected, LPS-challenged iMAECs. Both pScr and pA3p express GFP. Data points indicate two independent treatments with three biological replicates per respective treatment and bars are group means. ( D ) MiR-33a-3p expression measured via RT-qPCR in LPS-challenged iMAECs transfected with either pScr (Scr) or pA3p (A3p). Data points indicate three independent experiments with three biological replicates per respective experiment. Bars are group means and AU indicates arbitrary units.

Article Snippet: To measure mRNA expression of ABCA1 and Niemann–Pick type C1 (NPC1), we first converted total RNA extracted from cultured iMAECs into cDNA via using a qScript cDNA Ultra SuperMix Kit (Quantabio, Beverly, MA, USA) [ ].

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transgenic Assay, Transfection, Positive Control, Control, Flow Cytometry, Quantitative RT-PCR