Beas2B (ATCC, Manassas, VA) were grown in BEGM (Lonza, Basel, Switzerland). A549 cells and A549 cells with Smad4 deletion (Sigma-Aldrich, St. Louis, MO) were grown in F12 supplemented with 10% FBS and 2mM L-glutamine. H1299 cells (ATCC) were grown in RPMI supplemented with 10% FBS. Cell lines with stable Smad4 knockdown were created by transfecting cells with pcPUR+U6 containing an anti-Smad4 shRNA (52 (link)) (kindly provided by Hideaki Ijichi, University of Tokyo, Japan) followed by puromycin selection; the pcPUR+U6 cassette (iGene Therapeutics, Tokyo, Japan) was used as a control. Reduced Smad4 expression was confirmed by RT-qPCR and western blot against Smad4 (1:200 #7966 Santa Cruz) with GAPDH control (1:40000, ab8245, Abcam, Cambridge, MA). Cell line identity was validated by DNA profiling at the University of Colorado DNA Sequencing & Analysis Core.
Protocol detail
Smad4 Knockdown in Cell Lines
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A549 cells
Cell line
Cells
Deletion
Dna sequencing analysis
Gapdh
Glutamine
Puromycin
Shrna
Smad4
Therapeutics
Western blot
Corresponding Organization :
Other organizations : University of Colorado Anschutz Medical Campus, University of Colorado Denver
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Variable analysis
independent variables
- Smad4 knockdown in cell lines
- Smad4 expression (measured by RT-qPCR and western blot)
- Cell line identity (validated by DNA profiling)
- Cell culture media (BEGM, F12 + 10% FBS + 2mM L-glutamine, RPMI + 10% FBS)
- Cell lines (Beas2B, A549, A549 with Smad4 deletion, H1299)
- Positive control: pcPUR+U6 cassette (used as a control for Smad4 knockdown cell lines)
- Negative control: Not explicitly mentioned
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