Transient Transfection of siRNA and Plasmids

For transient transfection of siRNA and DNA plasmids, cells were seeded on day 1 in Nunclon^TM Delta Surface plates (Nunc, Roskilde, Denmark) and transfected twice, on days 2 and 3, serum-free with 25 or 50 nM of siRNA specific for RAC1B, PAR2, or ALK5 [8 (link),9 (link)], or the respective scrambled control siRNAs, for 4 h using Lipofectamine 2000 (Life Technologies). Additional validation of these siRNAs was performed for RAC1B [2 (link)], and ALK5 and PAR2 [8 (link),9 (link)]. A PAR2 expression vector (PAR2-Myc-DKK) was purchased from Origene (Rockville, MD, USA).

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Otterbein H., Mihara K., Hollenberg M.D., Lehnert H., Witte D, & Ungefroren H. (2019). RAC1B Suppresses TGF-β-Dependent Chemokinesis and Growth Inhibition through an Autoregulatory Feed-Forward Loop Involving PAR2 and ALK5. Cancers, 11(8), 1211.

Publication 2019

NunclonTM Delta Surface plates Lipofectamine 2000

Alk5 Cells Lipofectamine 2000 Plasmids Serum Sirnas Transfection Transient Vector

Corresponding Organization : University Hospital Schleswig-Holstein

Other organizations : University of Calgary

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Variable analysis

independent variables

SiRNA specific for RAC1B, PAR2, or ALK5
Respective scrambled control siRNAs
PAR2 expression vector (PAR2-Myc-DKK)

dependent variables

Not explicitly mentioned

control variables

Cells seeded on Nunclon™ Delta Surface plates
Transfection performed in serum-free conditions

controls

Positive control: Respective scrambled control siRNAs
Negative control: Not explicitly mentioned

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