Silencing SMAD and ALK-5 in Glioma Cells

To silence the expression of SMAD2, SMAD3, SMAD4, or ALK-5, GIC were transiently transfected by electroporation (Neon transfection system, Invitrogen) using siRNA pools (ON-TARGET plus human SMAD2 siRNA-SMART pool L-003561-00, ON-TARGET plus human SMAD3 siRNA-SMART pool L-020067-00-0020, ON-TARGET plus human SMAD4 siRNA- SMART pool L-003902-00 and ON-TARGET plus human ALK-5 siRNA- SMART pool L-003929-00 (Dharmacon, Lafayette, CO). Non-targeting siRNA pool was used as a negative control. Lentiviral pGIPZ vectors encoding c-MET-specific (Oligo ID V3LHS_642486) or non-silencing control shRNA (Oligo ID RHS4346) were purchased from Thermo Scientific (Waltham, MA). Glioma cells were transduced with lentiviral particles produced as described^{37 (link)}. Stably transduced clones were isolated with 4 µg/ml puromycin and subjected to analyses and assays after 1–5 passage post selection.

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Papa E., Weller M., Weiss T., Ventura E., Burghardt I, & Szabó E. (2017). Negative control of the HGF/c-MET pathway by TGF-β: a new look at the regulation of stemness in glioblastoma. Cell Death & Disease, 8(12), 3210.

Publication 2017

Neon transfection system Lentiviral pGIPZ vectors

Alk 5 Assays C met Cells Clones Electroporation Glioma Human Neon Oligo Puromycin Shrna Sirna Smad2 Smad3 Smad4 Transfection Vectors

Corresponding Organization :

Other organizations : University Hospital of Zurich, University of Zurich

Top 5 similar protocols

Variable analysis

independent variables

Silencing of SMAD2, SMAD3, SMAD4, or ALK-5 using siRNA pools
Overexpression of c-MET using lentiviral pGIPZ vectors

dependent variables

Not explicitly mentioned

control variables

Non-targeting siRNA pool as a negative control
Non-silencing control shRNA in lentiviral pGIPZ vectors

controls

Positive control: Non-targeting siRNA pool
Negative control: Non-silencing control shRNA in lentiviral pGIPZ vectors

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