Cells were lysed at 4 C with glass beads in Lysis buf fer containing a protease

Intracellular protec tive factors expressed in differentiated cells would not be secreted into the media, and would therefore not be expected to protect undifferentiatedna order GlcNAcstatin ve cells. To determine whether the protective effect of RATPA mediated differentiation is derived from cell autonomous or non cell autonomous factors, we performed dose response survival assays on na ve neuroblastoma cells in culture for 24 hours with different concentrations of 6 OHDA diluted in fresh media or six day conditioned media from cells treated with Neurobasal A media containing FBS, RA or RATPA, A protective effect of 1. 4 to 1. 6 fold, which was more pronounced in SK N SH cells, was observed for RA or RATPA conditioned media over fresh media of the same type, while treatment in conditioned media from cells grown in FBS actually decreased survival compared Organism to fresh media containing FBS, These data suggest that secreted factors present in the conditioned media from differentiated cells may play a role in protection from 6 OHDA toxicity. Identification of Differentially Expressed Genes in Differentiated Neuroblastoma Cells Because the protective effect of differentiation can be recapit ulated in two separate neuroblastoma cell lines, we reasoned that comparison of gene expression between undifferentiated and differentiated cells in both lines would allow us to narrow the list of potential neuroprotective factors over either cell line individually. Because protection against 6 OHDA toxicity was more pro nounced in RATPA treated cells, we chose to compare cells in this condition to undifferentiated cells cultured in FBS for gene expression analysis. Gene expression analysis was performed using two color hybridization to Agilent 44K microarrays, which permits normalization of gene expression signals from each cell condition to a universal human reference expression library, The difference between normalized log ratio buy BMS-911543 values for each gene on the array was calculated for undifferentiated versus differen tiated conditions in each cell line, and is displayed as a comparison between SH SY5Y and SK N SH cells, Positive values indicate genes whose expression is up regulated during differen tiation, while negative values indicate genes that are down regulated. The most differentially expressed genes common to both cell lines are displayed with their relative log fold change, Top hits on this list include genes known to be modulated during RA mediated neuronal differentiation, includ ing CYP26A1 and MMP9, To ensure that the microarray data accurately detected changes in gene expression, we performed quantitative RT PCR on five genes whose expression changed during RATPA mediated differentiation.