Neuroblast Differentiation and Lentiviral Transduction

Undifferentiated PC12 cells were plated onto collagen IV-coated plates (Corning) and differentiated in culture medium containing 50 ng/ml NGF (Accurate Chemical) for 5 – 7 days. Primary sympathetic neuroblasts isolated from SCGs of post-gestation rat pups (P2) were cultured as previously described [20 (link)]. Differentiated PC12 cells and neuroblasts were infected with lentivirus encoding shXAF1 or nontargeting control virus (shSCR) followed by NGF withdrawal with anti-NGF antibody (1:5000, Sigma) for up to 48 hours where they were harvested for subsequent analysis.

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Choo Z., Lin Koh R.Y., Wallis K., Wei Koh T.J., Kuick C.H., Sobrado V., Kenchappa R.S., Pheng Loh A.H., Soh S.Y., Schlisio S., En Chang K.T, & Chen Z.X. (2016). XAF1 promotes neuroblastoma tumor suppression and is required for KIF1Bβ-mediated apoptosis. Oncotarget, 7(23), 34229-34239.

Publication 2016

collagen IV-coated plates NGF anti-NGF antibody

Anti antibody Collagen iv Culture medium Lentivirus Pc12 cells Scgs Virus

Corresponding Organization :

Other organizations : National University of Singapore, Ludwig Cancer Research, Karolinska Institutet, Ngee Ann Polytechnic, KK Women's and Children's Hospital, Moffitt Cancer Center

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Variable analysis

independent variables

Lentivirus encoding shXAF1 or nontargeting control virus (shSCR)
NGF withdrawal with anti-NGF antibody (1:5000, Sigma)

dependent variables

Harvested cells for subsequent analysis

control variables

Undifferentiated PC12 cells plated onto collagen IV-coated plates (Corning)
Differentiation of PC12 cells in culture medium containing 50 ng/ml NGF (Accurate Chemical) for 5 – 7 days
Primary sympathetic neuroblasts isolated from SCGs of post-gestation rat pups (P2) and cultured as previously described [20]

positive controls

Differentiated PC12 cells
Primary sympathetic neuroblasts

negative controls

Nontargeting control virus (shSCR)

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