Figure 2. Mechanisms of increased GPC2 expression in neuroblastoma.

(A) GPC2 expression in two neuroblastoma tumor data sets stratified by chromosome 7q/GPC2 locus gain and MYCN amplification status (left, RNA sequencing, n = 64; right, mRNA microarray, n = 118; TARGET).

(B) MYCN ChIP plot showing MYCN binds an Ebox motif upstream of the GPC2 promoter in the MYCN amplified neuroblastoma cell lines Kelly, NGP, and NB-1643. Arrow head indicates location of Ebox motif (CACGTG).

(C) GPC2 reporter assay with and without forced overexpression of MYCN in SHEPneuroblastoma and HEK293T cells. Inset with Western blot displaying MYCN overexpression incorresponding cells.

(D) GPC2 quantitative PCR in 4-oHT MYCN inducible SKNAS neuroblastoma cells (SKNAS-NmycER) ± 4-OHT.

(E) MYCN/GPC2 quantitative PCR in the MYCN amplified neuroblastoma cell line Kelly after MYCN depletion with 2 unique shRNAs, shMYCN-5 and 6.

(F) Western blot of MYCN and GPC2 in the MYCN amplified neuroblastoma cell lines Kelly and NGP after MYCN depletion with an expanded set of 4 unique shRNAs, shMYCN-1, 5, 6 and 7.

Horizontal lines in A indicate mean ± SEM. Data in C are represented as mean ± SEM of at least 3 biological replicates. Data in D and E are represented as mean ± SEM of one experiment. Each experiment was repeated 2-4 times with similar results.

*p < 0.05; **p < 0.01; ***p < 0.0001; ns, not significant were derived via unpaired t tests.

Amp, amplification; Empty, pLenti CMV Puro empty vector; MYCN, MYCN pLenti CMV Puro vector; hr, hour. shNTC, non-targeting control shRNA.