Endothelial Cell Flow Mechanics and Gene Silencing

Human umbilical vein EC and porcine aortic EC were isolated and cultured.^{32 (link)} Gene silencing was performed using 2 different small interfering RNAs (siRNAs) against TWIST1 ((Silencer Select S14523, Ambion, and L-006434-00-0005 ON-TARGETplus SMARTpool; Dharmacon) or GATA4 (Silencer Select s5603, Ambion, and L-008244-00-0005 ON-TARGETplus SMARTpool; Dharmacon). They were exposed to flow using an orbital shaking system or Ibidi parallel-plate system.^{32 (link),33 (link)} Quantitative RT-PCR, immunofluorescent staining and chromatin immunoprecipitation,^{6 (link)} and assays of permeability^{34 (link)} were performed as described.

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Mahmoud M.M., Kim H.R., Xing R., Hsiao S., Mammoto A., Chen J., Serbanovic-Canic J., Feng S., Bowden N.P., Maguire R., Ariaans M., Francis S.E., Weinberg P.D., van der Heiden K., Jones E.A., Chico T.J., Ridger V, & Evans P.C. (2016). TWIST1 Integrates Endothelial Responses to Flow in Vascular Dysfunction and Atherosclerosis. Circulation Research, 119(3), 450-462.

Publication 2016

ON-TARGETplus SMARTpool

Aortic Assays Chromatin immunoprecipitation Human Immunofluorescent Porcine Rt pcr Sirnas Twist1 Umbilical vein

Corresponding Organization : KU Leuven

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Protocol cited in 8 other protocols

Variable analysis

independent variables

TWIST1 gene silencing using 2 different small interfering RNAs (siRNAs)
GATA4 gene silencing using 2 different small interfering RNAs (siRNAs)

dependent variables

Gene expression (quantified by quantitative RT-PCR)
Protein expression (assessed by immunofluorescent staining)
Protein-DNA interactions (measured by chromatin immunoprecipitation)
Permeability (assayed)

control variables

Human umbilical vein endothelial cells (EC) and porcine aortic EC
Exposure to flow using an orbital shaking system or Ibidi parallel-plate system

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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