Fig. 1.

MYCN amplification is associated with high Chk1 signaling and RS. a) Total RNA extracted from MYCN-amplified NB cell lines (blue: SK-N-DZ, SK-N-BE(2)c, BE(2)c, CHP-212, and IMR-32), non-MYCN-amplified NB cell lines (green: SK-N-AS, SK-N-SH, and SH-SY5Y), and non-malignant human cell lines (red: HCN1-A and hBM-MSCs) were analyzed by microarray analysis. The Log2 transformed Chk1 (X-axis) and MYCN (Y-axis) mRNA levels in these cell lines were graphed. The correlation between relative Chk1 and MYCN mRNA levels in these cell lines were analyzed by linear regression (r = 0.886). b) Total cell extracts from the indicated cell lines were analyzed by Western blotting to determine the levels of p-Chk1, Chk1, and Rad17. c) A microarray dataset of NB patient specimens (ArrayExpress: E-MTAB-179) was analyzed. The relative expression of Chk1 mRNA in patient specimens with or without MYCN amplification was graphed with the bottom and top ends of the whiskers representing the 5th and 95th percentiles of the expression levels respectively. “All” represents all non-MYCN-amplified NB samples. The “low-risk” subset includes patients with stage 1, 2, or 4S non-MYCN-amplified NB and under the age of 18 months. The “high-risk” subset includes patients with stage 4 non-MYCN-amplified tumors and above the age of 18 months. The p-value was determined by an unpaired t-test. Serial sections made from d) patient NB specimens and e) PTX NB tumors were stained with the indicated antibodies, as described in the Materials and Methods. The left two columns in each panel box are serial sections made from two MYCN-amplified tumors and the right two columns are sections made from two non-MYCN-amplified tumors.