Schematic drawing of the dox-regulatable VEGF-A shRNA lentivirus vector.

<p>Lentivirus vector for inducible knockdown permits constitutive expression of a Tet-transactivating component (rtTA3) with a Venus selection marker (green fluorescent protein -like protein) and tetracycline-regulated expression of VEGF-A-shRNAs. The shRNA transcripts were designed as primary microRNA mimics i.e. they were embedded in the primary transcript of human miRNA30. The lentiviruses were self-inactivating (SIN) third generation vectors, in which part of the viral 3´LTR has been deleted preventing the viral replication. The vectors contain a central polypurine tract (indicated as FLAP) for enhancement of viral titers and a woodchuck hepatitis virus posttranscriptional regulatory element (WRE) for better transgene expression [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190981#pone.0190981.ref024" target="_blank">24</a>].</p

Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice - Fig 3

<p><b>Plasma VEGF-A concentration in response to dox treatment (a). Tissue VEGF-A mRNA and protein levels after two weeks of 1 mg/ml dox treatment and five weeks of 2 mg/ml dox dose treatment (b-k).</b> Dox treatment decreased plasma VEGF-A levels in TG mice after 1 week, after which the plasma VEGF-A increased. In WT mice a decreasing effect on plasma VEGF-A was seen (a). *p<0.05, **p<0.01 and ***p<0.001 compared to baseline within each group, 1-way ANOVA with Dunnett´s post hoc test, n = 9-10/group. In the selected tissues, aorta, heart and kidney, the dox treatment with the 1 mg/ml dox dose for 2 weeks showed decreasing trend in VEGF-A mRNA expression in TG mice in comparison to WT mice (b, d, h), which was associated with increased cardiac VEGF-A protein (e) and decreased kidney VEGF-A protein levels (i). When the dox dose was doubled and the treatment time increased to 5 weeks, a trend towards increasing VEGF-A expression was seen in all three tissues in both WT and TG mice (c, f, j). However, no changes were detected in protein levels (g, k). **p<0.01 and ***p<0.001 compared to WT+dox group in 2 weeks experiment (b, d, e, h, i) or to no dox group (-dox) in 5 weeks experiment (c, f, g, j, k), <i>t-test</i>, n = 6-24/group.</p

VEGF-A knockdown via RNAi in mouse endothelial cells and cardiomyocytes in a doxycycline-regulatable fashion.

<p>Secreted VEGF-A protein amount was most efficiently decreased with sh1 -knockdown vector (T-1 and TT-1) in mouse endothelial cells in comparison to sh2 (T-2 and TT-2) and sh3 (T-3 and TT-3) vectors. With the sh1 construct the normal TRE promoter (T-1) seemed to be slightly more efficient and less leaky than the tight TRE promoter (TT-1) (a). The magnitude of VEGF-A knockdown with the selected T-1 vector increased with increasing dox doses in endothelial cells (b). With the increasing dox doses also the amount of Venus expressing cells (%) was increased as follows: 40 ± 7% (–dox), 46 ± 8% (dox 10 ng/ml), 85 ± 3% (dox 100 ng/ml) and 78 ± 2% (dox 1000 ng/ml) (c). In cardiomyocytes the knockdown of VEGF-A with the T-1 vector was shown to be dependent of the amount of virus (MOI) and dox exposure time and the decrease was larger at the cellular level in comparison to the secreted protein (d-e). Results are shown as mean ± S.D., n = 3/group. The percentage of VEGF-A protein concentration compared to control +dox group (a) or non-transduced (NT) group (b, d, e) are shown above bars. *p<0.05, **p<0.01 and ***p<0.001 compared to control +dox group (a) or non-transduced (NT) group (b, d, e), 1-way ANOVA with Dunnett´s post hoc test. NT = non-transduced cells, MOI = multiplicity of infection, Contr = Control lentivirus vector targeting Luciferase. Scale bar 200 μm (c).</p

dvdres-sep-2017-00131-File003 – Supplemental material for <i>In vivo</i> inhibition of nuclear factor of activated T-cells leads to atherosclerotic plaque regression in IGF-II/LDLR^–/–ApoB^100/100 mice

<p>Supplemental material, dvdres-sep-2017-00131-File003 for <i>In vivo</i> inhibition of nuclear factor of activated T-cells leads to atherosclerotic plaque regression in IGF-II/LDLR^–/–ApoB^100/100 mice by Fabiana Blanco, Suvi E Heinonen, Erika Gurzeler, Lisa M Berglund, Anna-Maria Dutius Andersson, Olga Kotova, Ann-Cathrine Jönsson-Rylander, Seppo Ylä-Herttuala and Maria F Gomez in Diabetes & Vascular Disease Research</p

dvdres-sep-2017-00131-File004 – Supplemental material for <i>In vivo</i> inhibition of nuclear factor of activated T-cells leads to atherosclerotic plaque regression in IGF-II/LDLR^–/–ApoB^100/100 mice

<p>Supplemental material, dvdres-sep-2017-00131-File004 for <i>In vivo</i> inhibition of nuclear factor of activated T-cells leads to atherosclerotic plaque regression in IGF-II/LDLR^–/–ApoB^100/100 mice by Fabiana Blanco, Suvi E Heinonen, Erika Gurzeler, Lisa M Berglund, Anna-Maria Dutius Andersson, Olga Kotova, Ann-Cathrine Jönsson-Rylander, Seppo Ylä-Herttuala and Maria F Gomez in Diabetes & Vascular Disease Research</p