Journal: PLoS ONE

Article Title: Acetate Supplementation Induces Growth Arrest of NG2/PDGFRα-Positive Oligodendroglioma-Derived Tumor-Initiating Cells

doi: 10.1371/journal.pone.0080714

Figure Lengend Snippet: OG33 (A) and OG35 (B-D) cells were induced to differentiate over 6 days in vitro via activation of adenylyl cyclase (1 mM dibutyryl cAMP or 10 μM forskolin) or inhibition of MEK1/2 (1 μM PD035901), ErbB2 (1 μM PD174285), PI3K (5 μM and 10 μM LY294002), or mTOR (10 nM and 20 nM rapamycin [RAP]) signaling. DMSO (0.2% DMSO) was used as a control for PD174285, PD035901 and LY294002 treatments. Despite inhibition of PI3K-Akt-mTOR and ERK signaling pathways, both cells failed to differentiate into cells with oligodendroglial morphology (A, B) or increase immunoreactivity for GPAF or CNPase (not shown). MEK and ErbB2 inhibition actually increased OG35 proliferation in DM. While inhibition of PI3K-Akt-mTOR signaling induced morphological alterations in OG35 cells (B), this was not associated with increased CNPase (C) or ASPA (D) protein levels. In fact, ASPA expression decreased in the presence of 10 μM LY294002 and 20 nM rapamycin. Differentiation of Oli-Neu cells with cAMP for 4 days or inhibition of ErbB2 signaling for 2 days served as controls for oligodendrocyte cell morphology. DM - differentiation medium, SCM - stem cell medium. n = 3 independent cultures. *p < 0.05. Scale bar = 100 μm.

Article Snippet: For pharmacological induction of differentiation, OG33 and OG35 cells were plated in SCM or DM in the absence or presence of dibutyryl cAMP (1 mM in water, Sigma), forskolin (10 μM in ethanol, Sigma), the MEK1/2 inhibitor PD035901 (1 μM in dimethylsulfoxide [DMSO], Tocris Bioscience/R & D Systems; Minneapolis, MN), the ErbB2 inhibitor PD174285 (1 μM in DMSO, Santa Cruz Biotechnology), the PI3K inhibitor LY294002 (at 5 μM and 10 μM in DMSO, Tocris), the mTOR inhibitor rapamycin (at 10 nM and 20 nM in ethanol, Sigma) or 0.2% DMSO as a control (final DMSO concentration of 0.05% for PD174285, 0.1% for PD035901 and LY294002, and 0.2% for PD035901/LY294002/rapamycin treatment).

Techniques: In Vitro, Activation Assay, Inhibition, Control, Protein-Protein interactions, Expressing